Chimeric protein comprising non-toxic pseudomonas exotoxin and type IV pilin sequences

ABSTRACT

The invention provides chimeric proteins comprising a non-toxic  Pseudomonas  exotoxin A sequence and a Type IV pilin loop sequence, wherein the Type IV loop sequence is inserted within the non-toxic  Pseudomonas  exotoxin A. The invention also provides polynucleotides encoding the chimeric proteins, and compositions comprising the polynucleotides or the chimeric proteins. The invention also provides methods for using the chimeric proteins, polynucleotides and compositions of the invention.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application No.60/257,877 filed Dec. 21, 2000, the contents of which is incorporatedherein by reference in their entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not Applicable.

BACKGROUND OF THE INVENTION

Type IV pilin is the major subunit of the pilus or pili which arefilamentous structures covering many microorganisms including bacteriaand yeast. Among these microorganisms, many pathogenic species expressType IV pilins, including, e.g., P. aeruginosa, N. meningitides, N.gonorrhoeae, Vibro cholera, and Pasteurella multocidam. The first stepin infection with these pathogenic microorganisms is adherence to targetcells through the pili. In particular, Type IV pilins of Pseudomonasaeruginosa bind to asialoGM1 receptors on epithelial cells (Saiman etal., J. Clin. Invest. 92(4):1875-80 (1993); Sheth et al., 11 (4):715-23(1994); Imundo et al., Proc. Natl. Acad. Sci. USA, 92(7):3019-23 (1995);Hahn, Gene 192(1):99-108 (1997)). Thus, the pili of these microorganismsare a major virulence factor, and result in colonization by pathogenicmicroorganisms and infections in humans.

For example, Pseudomonas aeruginosa causes between 10% and 20%infections in most hospitals. Pseudomonas infection is common amongpatients with cystic fibrosis, burn wounds, organ transplants, andintravenous-drug addiction. Pseudomonas infections can lead to seriousconditions, such as endophthalmitis, endocarditis, meningitis,pneumonia, and septicemia. In particular, colonization of cysticfibrosis (CF) individuals with Pseudomonas aeruginosa represents asignificant negative milestone in the progression of this disease. Oncecolonized, patients are subject to the damaging effects of varioussecreted virulence factors and to the inflammatory response of the hostimmune system.

Type IV pili are composed of pilin polymers arranged in a helicalstructure with five subunits per turn (Forest et al., Gene 192(1):165-9(1997); Parge, Nature 378(6552):32-8 (1995)). The portion of the pilinprotein responsible for cell binding is found near the C-terminus (aminoacids 122-148) in a β-turn loop subtended from a disulfide bond(Campbell et al., Biochemistry 36(42):12791-801 (1997); Campbell et al.,J. Mol. Biol. 267(2):382-402 (1997); Hazes et al., J. Mol. Biol.299(4):1005-1017 (2000); McInnes et al., Biochemistry 32(49):13432-40(1993)). For P. aeruginosa, a 12 or 17 amino acid sequence (depending onthe strain) in this loop interacts with receptors on epithelial cells.For CF individuals, the overproduction of the R domain of mutant cysticfibrosis transmembrane conductance regulator (CFTR) can lead to anincreased level of asialoGM1 and, accordingly, an increased binding ofP. aeruginosa (Imundo et al., Proc. Natl. Acad. Sci. USA 92(7):3019-23(1995); Saiman et al., J. Clin. Invest. 92(4):1875-80 (1993); Bryan etal., Am. J. Respir. Cell Mol. Biol. 19(2):269-77 (1998); Imundo et al.,Proc. Natl. Acad. Sci. USA 92(7):3019-23 (1995); Saiman et al., J. Clin.Invest. 92(4):1875-80 (1993)). Functional studies of pilin haveindicated that only the last pilin subunit (the tip) of a pilusinteracts with epithelial cell receptors (Lee et al., Mol. Microbiol.11(4):705-13 (1994)).

To date, efforts to produce an effective anti-pilin vaccine have notbeen very successful. In part, this limited success is because the mostimmunogenic portion of the protein (the middle) does not generateantibodies that interfere with adhesion. Unfortunately, the C-terminalloop of pilin is not very immunogenic, and high titer responses haveonly been reported with the use of strategies that employ multipledisplay copies of the loop sequence (Hahn et al., Behring. Inst. Mitt.(98):315-25 (1997)). For CF patients, strategies to inhibit Pseudomonascolonization are considered an important element in reducing themorbidity normally associated with the development of chronic infections(Tang et al., Infect. Immun. 63(4):1278-85 (1995); Li et al., Proc.Natl. Acad. Sci. USA 94(3):967-72 (1997); Tang et al., Infect. Immun.63(4):1278-85 (1995) Doig, P. et al., Infect Immun 58(1):124-30 (1990);El-Zaim, H. S. et al. Infect Immun 66(11):5551-4 (1998)).

Accordingly, there is a need to develop compositions for reducing orpreventing infections by pathogenic microorganisms including, inparticular, Pseudomonas aeruginosa. Embodiments of this inventionaddress this and other needs.

SUMMARY OF THE INVENTION

Embodiments of the invention provide chimeric proteins comprising anon-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loopsequence, wherein the Type IV pilin loop sequence is located within thenon-toxic Pseudomonas exotoxin A sequence. In the present invention, aType IV pilin loop sequence refers to the sequence that forms anintrachain disulfide loop at the C-terminus of the pilin. This loopinteracts and binds to receptors on epithelial cells. The presentinvention is based on, in part, the discovery that the Type IV pilinloop sequence within the Pseudomonas exotoxin A sequence is presented innear-native conformation, and can react with receptors on epithelialcells. As a result, the present chimeric protein comprises the Type IVpilin loop sequence which competes for binding to these epithelialcells, and which can reduce adherence of pathogenic microorganismsexpressing the Type IV pilin to the epithelial cells. Therefore, thechimeric protein can be used on its own or in a composition to directlyreduce adherence of pathogenic microorganisms in a host.

The present invention is also based on, in part, the discovery thatantisera generated against the chimeric proteins of the invention arealso useful in reducing adherence of pathogenic microorganisms(expressing Type IV pilins) in a host. Since the chimeric proteinpresents the Type IV pilin loop in near-native conformation, thechimeric proteins of the invention, when introduced into a host,generate polyclonal antisera that bind to the pilin loop portion of thechimeric proteins. The antisera can also bind to Type IV pilins onpathogenic microorganism, and thus competitively inhibit binding of thepathogenic microorganisms to epithelial cell receptors. Accordingly, thechimeric protein can be used as a vaccine to generate antisera in a hostwhich can result in reduction of both adherence and colonization ofpathogenic microorganisms in the host.

Furthermore, since the chimeric protein presents the non-toxicPseudomonas exotoxin A sequence in near-native conformation, thechimeric proteins of the invention, when introduced into a host,generate polyclonal antisera that bind to the non-toxic Pseudomonasexotoxin A as well as to the native Pseudomonas exotoxin A. The nativePseudomonas exotoxin A which is secreted by Pseudomonas aeruginosa isknown to cause cell cytotoxicity by entering into cells byreceptor-mediated endocytosis and then, after a series of intracellularprocessing steps, translocate to the cell cytosol and ADP-ribosylateelongation factor 2. This results in the inhibition of protein synthesisand cell death. The antisera generated against the present chimericprotein can bind exotoxin A released from Pseudomonas and can neutralizecell cytotoxicity. Therefore, should small numbers of Pseudomonasovercome the first line of defense (antibodies against the pilin loopsequence preventing colonization), the normal destructive power of theexotoxin A will be neutralized by antibodies generated against thenon-toxic Pseudomonas exotoxin A sequence.

The chimeric proteins, the chimeric polynucleotides, and thecompositions of the present invention have many other utilities. Forexample, the chimeric proteins and the compositions comprising chimericproteins can be used to in diagnostic tests, such as immunoassays. Suchdiagnostic tests can be used to detect the presence of microorganismsbearing a Type IV pilin loop sequence, such as Pseudomonas aeruginosa,or to determine whether a host has antisera against a Type IV pilin loopdue to an infection. In another example, the chimeric proteins and thecompositions comprising the chimeric proteins can also be used to purifyantibodies against, e.g., the Type IV pilin loop sequence. In anotherexample, the antibodies against the chimeric protein can be used toclone and isolate other related Type IV pilin sequences.

Accordingly, in one aspect of the invention, the invention provides achimeric protein comprising: a non-toxic Pseudomonas exotoxin A sequenceand a Type IV pilin loop sequence, the Type IV pilin loop sequence beinglocated within the non-toxic Pseudomonas exotoxin A sequence, whereinthe chimeric protein is capable of reducing adherence of a microorganismexpressing the Type IV pilin loop sequence to epithelial cells, andfurther wherein the chimeric protein, when introduced into a host, iscapable of generating polyclonal antisera that reduce adherence of themicroorganism expressing the Type IV pilin loop sequence to theepithelial cells.

In another aspect, the invention provides a chimeric protein comprising:(a) a non-toxic Pseudomonas exotoxin A sequence comprising domain Ia,domain II, and domain III; and (b) a Type IV pilin loop sequence,wherein the Type IV pilin loop sequence is located between domain II anddomain III of the non-toxic Pseudomonas exotoxin A sequence.

In another aspect, the invention provides a polynucleotide encoding achimeric protein, the chimeric protein comprising: a non-toxicPseudomonas exotoxin A sequence and a Type IV pilin loop sequence, theType IV pilin loop sequence being located within the non-toxicPseudomonas exotoxin A sequence, wherein the chimeric protein is capableof reducing adherence of a microorganism expressing the Type IV pilinloop sequence to epithelial cells, and ftuther wherein the chimericprotein, when introduced into a host, is capable of generatingpolyclonal antisera that prevent adherence of the microorganismexpressing the Type IV pilin loop sequence to the epithelial cells.

In another aspect, the invention provides a polynucleotide encoding achimeric protein, the chimeric protein comprising: (a) a non-toxicPseudomonas exotoxin A sequence comprising domain Ia, domain II, anddomain III; and (b) a Type IV pilin loop sequence, wherein the Type IVpilin loop sequence is located between domain II and domain III of thenon-toxic Pseudomonas exotoxin A sequence.

In another aspect, the invention provides a composition comprising achimeric protein, the chimeric protein comprising: a non-toxicPseudomonas exotoxin A sequence and a Type IV pilin loop sequence, theType IV pilin loop sequence being located within the non-toxicPseudomonas exotoxin A sequence, wherein the chimeric protein is capableof reducing adherence of a microorganism expressing the Type IV pilinloop sequence to epithelial cells, and further wherein the chimericprotein, when introduced into a host, is capable of generatingpolyclonal antisera that prevent adherence of the microorganismexpressing the Type IV pilin loop sequence to the epithelial cells.

In another aspect, the invention provides a method for eliciting animmune response in a host, the method comprising the step ofadministering to the host an immunologically effective amount of acomposition comprising a chimeric protein comprising: a non-toxicPseudomonas exotoxin A sequence and a Type IV pilin loop sequence, theType IV pilin loop sequence being located within the non-toxicPseudomonas exotoxin A sequence, wherein the chimeric protein is capableof reducing adherence of a microorganism expressing the Type IV pilinloop sequence to epithelial cells, and further wherein the chimericprotein, when introduced into the host, is capable of generatingpolyclonal antisera that prevent adherence of the microorganismexpressing the Type IV pilin loop sequence to the epithelial cells.

In another aspect, the invention provides a method of eliciting animmune response in a host, the method comprising the step ofadministering to the host an immunologically effective amount of anexpression cassette comprising a polynucleotide encoding a chimericprotein comprising: a non-toxic Pseudomonas exotoxin A sequence and aType IV pilin loop sequence, the Type IV pilin loop sequence beinglocated within the non-toxic Pseudomonas exotoxin A, wherein thechimeric protein is capable of reducing adherence of a microorganismexpressing the Type IV pilin loop sequence to epithelial cells, andfurther wherein the chimeric protein, when introduced into the host, iscapable of generating polyclonal antisera that reduce adherence of themicroorganism expressing the Type IV pilin loop sequence to theepithelial cells.

In another aspect, the invention provides a method of generatingantibodies specific for a Type IV pilin loop sequence, comprisingintroducing into a host a composition comprising a chimeric proteincomprising a non-toxic Pseudomonas exotoxin A sequence and a Type IVpilin loop sequence, the Type IV pilin loop sequence being locatedwithin the non-toxic Pseudomonas exotoxin A, wherein the chimericprotein is capable of reducing adherence of a microorganism expressingthe Type IV pilin loop sequence to epithelial cells, and further whereinthe chimeric protein, when introduced into the host, is capable ofgenerating polyclonal antisera that reduce adherence of themicroorganism expressing the Type IV pilin loop sequence to epithelialcells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates in cartoon form the replacement of domain Ib withthe C-terminal loop of pilin. The pilin insert corresponds to thesequence of pilin reported for the PAK strain of P. aeruginosa.

FIG. 1B illustrates in cartoon form the domain structure of PE fromAllured et al., Proc. Natl. Acad. Sci. 83:1320-1324 (1986). PE64 lacksthe loop region of domain Ib. PE64pil includes the insertion of thepilin loop (residues 129-142) of the PAK strain of P. aeruginosa. Thedeletion of glutamic acid 553 (indicated by a dot) removes an activesite residue (Lukac et al., Infect. Immuno. 56(12):3095-8 (1988)) andproduces proteins PE64Δ553 and PE64Δ553pil with no ADP-ribosylatingactivity. The Ib loop is shown in light shading and the pilin loop indarker shading.

FIG. 2 illustrates SDS PAGE (Panel A and C) and Western blot analysis(Panel B) of PE proteins and pilin. A. Lanes 1-4 show substantially purePE proteins (4-5 μg of protein was loaded per lane) after MonoQchromatography. From left to right the proteins loaded were: PE64,PE64pil, PE64Δ553 and PE64Δ553pil. Purified PAK pilin was added to lane5. B. Lanes 6-10 show the same proteins as A but probed with amonoclonal antibody to the pilin loop. Lane 11 is PE64Δ553pil after gelfiltration chromatography. Standard proteins and their molecular massesin kDa are indicated.

FIG. 3 illustrates the toxicity of PE64pil compared to PE64. To assessthe effect of introducing a third party loop into PE, we compared thetoxicity of PE64 (▪) with PE64pil (▴). Increasing concentrations of eachprotein was added to L929 cells and, after an overnight incubation,inhibition of protein synthesis was determined. Results are expressed aspercent control compared to cells receiving no toxin. Error barsrepresent one SD of the mean from triplicate wells.

FIG. 4 illustrates the interaction of PE64pil and PE64Δ553pil withimmobilized asialo-GM1. (A). Various concentrations of PE64pil or PE64were added to plates coated with asialo-GM1 and binding was determinedby reactivity with rabbit anti-PE followed by a peroxidase labeled goatanti-rabbit IgG antibody. Absorbance at 450 μm was used to monitorbinding. (B). and (C). To investigate ganglioside specificity, acompetition assay was devised whereby soluble asialo-GM1 ormonosialo-GM1 at 2 ug/ml was preincubated with PE64pil (B) orPE64Δ553pil (C) and the percent residual binding determined as describedin panel (A). For (B) and (C), graphs show the mean of a representativetriplicate experiment. Error bars represent one SD. N.A.=no addition ofcompetitor.

FIG. 5 illustrates adhesion of Ps. aeruginosa (PAK strain) to A549cells. Bacteria were added to cells at an MOI of 100 in the presence orabsence of potential inhibitors. Peptides were added to a finalconcentration of 40 μM, while proteins were added to a concentration of2 μM. The graph indicates the percentage of cell-bound bacteria comparedto samples with no inhibitor. Error bars represent one standarddeviation from the mean of three independent experiments.

FIG. 6 illustrates antibody titers post immunization with PE64pil withand without adjuvant. Sera were collected from each of four rabbits(numbered 87-90) at various times, diluted 1:100 and then added tostreptavidin-coated plates that had been loaded with biotinylated pilinpeptides. Rabbit IgG was detected by the addition of a peroxidaseconjugated goat anti-rabbit antibody. Rabbits 87 and 88 receivedadjuvant while rabbits 89 and 90 did not.

FIG. 7 illustrates antibody-mediated interference with adhesion to A549cells. (A). The PAK strain of Ps. aeruginosa was incubated with 1:20 to1:100 dilutions of prebleed or immune (taken after the fourth injectionof antigen) sera from rabbit #87. Bacteria were then added to cells andthe percent adhesion determined by comparison with bacteria that hadbeen incubated in media alone. (B). A 1:20 dilution of sera from eachrabbit, prebleed and immune, was tested for antibody mediatedinterference. (C). Various strains of Ps. aeruginosa were incubated withimmune sera (1:20) from one of the rabbits that received antigen alone(rabbit #90) and one that received antigen plus adjuvant (rabbit #88).For each panel of FIG. 7, the bar represents the number of bacteria percell determined by examining one hundred A549 cells. The error barsrepresent one standard deviation from the mean of three independentexperiments.

FIG. 8 illustrates antibody-mediated neutralization of PE toxicity.Immune sera (▴) or prebleed sera (▪) were diluted 1:20 and mixed withPE64 at 1.0 ug/ml. Samples were then diluted to the concentrationindicated and added to L929 cells for an overnight incubation. Resultsare expressed as percent control of protein synthesis compared to cellsreceiving no toxin. Error bars represent one SD of the mean fromtriplicate wells.

DEFINITIONS

“Pseudomonas exotoxin A” or “PE” is secreted by P. aeruginosa as a 67kDa protein composed of three prominent globular domains (Ia, II, andIII) and one small subdomain (Ib) connecting domains II and III.(Allured et. al., Proc. Natl. Acad. Sci. 83:1320-1324 (1986).) Domain Iaof PE located at the N-terminus and mediates cell binding. In nature,domain Ia binds to the low density lipoprotein receptor-related protein(“LRP”), also known as the α2-macroglobulin receptor (“α2-MR”). (Kounnaset al., J. Biol. Chem. 267:12420-23 (1992).) It spans amino acids 1-252.Domain II mediates translocation to the cytosol. It spans amino acids253-364. Domain lb has no known function. It spans amino acids 365-399.Domain III is responsible for cytotoxicity and includes an endoplasmicreticulum retention sequence. It mediates ADP ribosylation of elongationfactor 2 (“EF2”), which inactivates protein synthesis. It spans aminoacids 400-613. The native Pseudomonas aeruginosa exotoxin A nucleic acidsequence and the amino acid sequence are shown as SEQ ID NO:1 and SEQ IDNO:2, respectively. SEQ ID NOS: 1 and 2 are the mature form of exotoxinA, wherein the signal sequence has been cleaved off. As a virulencefactor, PE can kill PMNs, macrophages and other elements of the immunesystem (Pollack et al., Infect. Immuno. 19(3):1092-6 (1978)).

As used herein, “Pseudomonas exotoxin A” or “PE” refer to those havingthe functions described above and includes the native Pseudomonasexotoxin A having the nucleic acid and amino acid sequences (as shown asSEQ ID NO:1 and SEQ ID NO:2, respectively) and also polymorphicvariants, alleles, mutants and interspecies homologs that: (1) haveabout 80% amino acid sequence identity, preferably about 85-90% aminoacid sequence identity to SEQ ID NO:2 over a window of about 25 aminoacids, preferably over a window of about 50-100 amino acids; (2) bind toantibodies raised against an immunogen comprising an amino acid sequenceof SEQ ID NO:2 and conservatively modified variants thereof; or (3)specifically hybridize (with a size of at least about 500, preferably atleast about 900 nucleotides) under stringent hybridization conditions toa sequence SEQ ID NO:1 and conservatively modified variants thereof. Forexample, genetically modified forms of PE are described in, e.g., Pastanet al., U.S. Pat. No. 5,602,095; Pastan et al., U.S. Pat. No. 5,512,658and Pastan et al., U.S. Pat. No. 5,458,878. Allelic forms of PE areincluded in this definition. See, e.g., Vasil et al., Infect. Immunol.52:538-48 (1986).

“Non-toxic Pseudomonas exotoxin A” or “non-toxic PE” refers to anyPseudomonas exotoxin A described herein (including modified variants)that lacks ADP ribosylation activity. The ribosylating activity of PE islocated between about amino acids 400 and 600 of PE. For example,deleting amino acid E553 (“ΔE553”) from domain III detoxifies themolecule. This detoxified PE is referred to as “PE ΔE553.” In anotherexample, substitution of histidine residue of PE at 426 with a tyrosineresidue also inactivates the ADP-ribosylation of PE (see Kessler &Galloway, J. Biol. Chem. 267:19107-11 (1992)). Other amino acids withindomain III can be modified by, e.g., deletion, substitution or additionof amino acid residues, to eliminate ADP ribosylation activity. DomainIII of non-toxic PE is sometimes referred to herein as “detoxifieddomain III.”

The term “a non-toxic Pseudomonas exotoxin A sequence” is usedgenerically to refer to either a nucleic acid sequence or an amino acidsequence of non-toxic Pseudomonas exotoxin A. As used herein, anon-toxic Pseudomonas exotoxin A sequence may be a full length sequenceor portion(s) of the full length sequence. Generally, a non-toxicPseudomonas exotoxin A sequence has one or more domains or portions ofdomains with certain biological activities of a non-toxic Pseudomonasexotoxin A, such as a cell recognition domain, a translocation domain,or an endoplasmic reticulum retention domain. For example, a non-toxicPseudomonas exotoxin A sequence may include only domain II anddetoxified domain III. In another example, a non-toxic Pseudomonasexotoxin A sequence may include only domain Ia, domain II, anddetoxified domain III. In another example, a non-toxic Pseudomonasexotoxin A sequence may include all of domains Ia, Ib, II, anddetoxified III. Therefore, a non-toxic Pseudomonas exotoxin A sequencemay be a contiguous sequence of the native Pseudomonas exotoxin A, or itcan be a sequence comprised of non-contiguous subsequences of the nativePseudomonas exotoxin A that lacks ADP ribosylation activity. While anon-toxic Pseudomonas exotoxin A sequence may be smaller contiguous ornon-contiguous portion(s) of the native PE, the numberings of the nativePE amino acid and nucleic acid sequences are used to refer to certainpositions within the non-toxic Pseudomonas exotoxin A sequence (e.g.,deletion of Glu at position 553).

A “chimeric protein” or a “chimeric polynucleotide” is an artificiallyconstructed protein or polynucleotide comprising heterologous amino acidsequences or heterologous nucleic acid sequences, respectively.

The term “heterologous” when used with reference to a protein or anucleic acid indicates that the protein or the nucleic acid comprisestwo or more sequences or subsequences which are not found in the samerelationship to each other in nature. For instance, the nucleic acid istypically recombinantly produced, having two or more sequences fromunrelated genes arranged to make a new functional nucleic acid. Forexample, in one embodiment, the nucleic acid has a promoter from onegene arranged to direct the expression of a coding sequence from adifferent gene. Thus, with reference to the coding sequence, thepromoter is heterologous. Similarly, a sequence from a Pseudomonasexotoxin A is heterologous with reference to a Type IV pilin loopsequence when the two sequences are placed in a relationship other thanthe naturally occurring relationship of the nucleic acids in the genome.

“Type IV pili” refers to filamentous structures covering manygram-negative bacteria, yeast and other microorganisms. The pili on thesurface of a microorganism adhere to epithelial cells. In particular,the pili of Pseudomonas or Candida bind to epithelial cells throughspecific interaction with asialoGM1 receptors. Type IV pili areprimarily composed of protein pilins, which are polymers arranged in ahelical bundle. For example, pili of Pseudomonas aeruginosa have anaverage length of 2.5 μm and consist of a single protein with amolecular mass of around 15,000 (Paranchych et al., Am. Soc. Microbio.343-351 (1990)).

The term “Type IV pilin” as used herein refer to pilins that contain aconserved amino terminal hydrophobic domain beginning with anamino-terminal phenylalanine that is methylated upon processing andsecretion of the pilin. Another characteristic feature of Type IV pilinsis that in the propilin form they contain similar six- or seven-aminoacid long leader peptides, which are much shorter than typical signalsequences. Type IV pilins are expressed by several bacterial genuses,including Neisseria, Moraxella, Bacteroides, Pasteurella andPseudomonas, E. coli, and yeast such as Candida. Species within thesegenuses which express Type IV pilins are, for example, P. aeruginosa, N.gonorrhoeae, N. meningtidis, Pasteurella multocida, M. bovis, B.nodosus. As used herein, the term “Type IV pilin” also includes the Tcppilin of Vibrio, (e.g., V. cholera), that is highly homologous to theType IV pilins of other genuses. Tcp pilin contains the characteristicamino-terminal hydrophobic domain as well as having a modifiedN-terminal amino acid that in this case may be a modified methioninebecause the Tcp pilin gene encodes a methionine residue at the positionwhere all the others encode a phenylalanine. Precursor TcpA contains amuch longer leader sequence than typical Type IV propilins but retainshomology in the region surrounding the processing site. Generally, apilin protein comprises a region at the N-terminus that is highlyconserved, with the rest of the protein containing moderately conservedand hypervariable regions (Paranachych et al., supra). A characteristicfeature of all pilins is an intrachain disulfide loop at the C-terminusof the pilin.

The amino acid sequences and nucleic acid sequences of Type IV pilins ofvarious microorganisms are known in the art. See, e.g., NCBI DatabaseAccession No. M14849, J02609 for Pseudomonas PAK strain; NCBI DatabaseAccession No. AAC60462 for Pseudomonas T2A strain; NCBI DatabaseAccession No. M11323 for Pseudomonas PAO strain; NCBI Database AccessionNo. P17837 for Pseudomonas CD strain; NCBI Database Accession No. B31105for Pseudomonas P1 strain; NCBI Database Accession No. Q53391 forPseudomonas KB7 strain; NCBI Database Accession No. AAC60461 forPseudomonas 577B strain; NCBI Database Accession No. A33105 forPseudomonas K122-4 strain; NCBI Database Accession Nos. Z49820, Z69262,and Z69261 for N. meningtidis; NCBI Database Accession Nos. X66144 andAF043648 for N. gonorrhoeae; NCBI Database Accession Nos. U09807 andX64098 for V. cholera; NCBI Database Accession No. AF154834 forPasteurella multocida.

A “Type IV pilin loop sequence” refers to the sequence that forms anintrachain disulfide loop at the C-terminus of the pilin. This region isphysically exposed at the tip of the pilus, and interacts withepithelial cell receptors. A Type IV pilin loop sequence as used hereincan refer to a sequence between the two cysteine residues that form anintrachain disulfide loop at the C-terminus of the pilin (i.e.,excluding the cysteine residues), or a sequence that includes bothcysteine residues and amino acids between the two cysteine residues.Depending on whether the site of insertion within non-toxic Pseudomonasexotoxin A sequences has cysteine residues, the Type IV pilin loopsequence with or without the flanking cysteine residues can be used tomake chimeric proteins of the invention. Examples of Type IV pilin loopsequence are shown as SEQ ID NOS: 3 to 20.

The term “immunogenic fragment thereof” or “immunogenic portion thereof”refers to a polypeptide comprising an epitope that is recognized bycytotoxic T lymphocytes, helper T lymphocytes or B cells.

“Polyclonal antisera” refers to sera comprising polyclonal antibodiesagainst an immunogen, which sera is obtained from a host immunized withthe immunogen (e.g., a chimeric protein of the present invention).

Polyclonal antisera that “reduce adherence” of a microorganismexpressing a Type IV pilin loop sequence refer to polyclonal antiserathat reduce adherence of the microorganism by about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or 100%, compared to a control. A control can bea prebleed or sera that is not exposed to the chimeric proteins of thepresent invention.

The term polyclonal antisera that “neutralize cytotoxicity” ofPseudomonas exotoxin A in the context of the present invention refer tothe ability of antisera to reduce the inhibition of protein synthesis byPseudomonas exotoxin A. Typically, polyclonal antisera can reduceinhibition of protein synthesis by Pseudomonas exotoxin A by at leastabout 30%, more typically at least about 50%, more typically at leastabout 80%, even more typically at least about 90%, 95%, or 99% comparedto a control. A control can be a prebleed or sera that is not exposed tothe chimeric proteins of the present invention.

“Nucleic acid” or “polynucleotide” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. The term encompasses nucleic acids containingknown nucleotide analogs or modified backbone residues or linkages,which are synthetic, naturally occurring, and non-naturally occurring,which have similar binding properties as the reference nucleic acid, andwhich are metabolized in a manner similar to the reference nucleotides.Examples of such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” apply to both amino acid and nucleicacid sequences. With respect to particular nucleic acid sequences,conservatively modified variants refer to those nucleic acids whichencode identical or essentially identical amino acid sequences, or wherethe nucleic acid does not encode an amino acid sequence, to essentiallyidentical sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenprotein. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, optionally 10 timesbackground hybridization. Exemplary stringent hybridization conditionscan be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42°C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and0.1% SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

An “expression cassette” refers to a polynucleotide molecule comprisingexpression control sequences operatively linked to coding sequence(s).

A “vector” is a replicon in which another polynucleotide segment isattached, so as to bring about the replication and/or expression of theattached segment.

“Control sequence” refers to polynucleotide sequences which arenecessary to effect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism; in prokaryotes, such control sequences generally includepromoter, ribosomal binding site, and terminators; in eukaryotes,generally, such control sequences include promoters, terminators and, insome instances, enhancers. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is necessary forexpression, and may also include additional components whose presence isadvantageous, for example, leader sequences.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A control sequence “operably linked” to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under conditions compatible with the control sequences.

A “ligand” is a compound that specifically binds to a target molecule.

A “receptor” is compound that specifically binds to a ligand.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)).Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized antibodies. Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)).

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to fusion proteins can be selected to obtain only thosepolyclonal antibodies that are specifically immunoreactive with fusionprotein and not with individual components of the fusion proteins. Thisselection may be achieved by subtracting out antibodies that cross-reactwith the individual antigens. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select antibodies specifically immunoreactive with a protein (see,e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity). Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an individual antigen or a portion thereof) or maycomprise a variant of such a sequence. Polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the biological activity of the encoded chimericprotein is not diminished, relative to a chimeric protein comprisingnative antigens. Variants preferably exhibit at least about 70%identity, more preferably at least about 80% identity and mostpreferably at least about 90% identity to a polynucleotide sequence thatencodes a native polypeptide or a portion thereof.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 70% identity, optionally 75%, 80%, 85%, 90%, or 95% identity overa specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecompliment of a test sequence. Optionally, the identity exists over aregion that is at least about 25 to about 50 amino acids or nucleotidesin length, or optionally over a region that is 75-100 amino acids ornucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 25 to 500, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987). The method used is similar to the method described byHiggins & Sharp, CABIOS 5:151-153 (1989). The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g. version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395 (1984)).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

“Immunogen” refers to an entity that induces antibody production in thehost animal.

“Vaccine” refers to an agent or composition containing an agenteffective to confer a therapeutic degree of immunity on an organismwhile causing only very low levels of morbidity or mortality. Vaccinesand methods for making vaccines are useful in the study of the immunesystem and in preventing and treating animal or human disease.

An “immunogenic amount” or “immunologically effective amount” is anamount effective to elicit an immune response in a subject.

“Substantially pure” or “isolated” means an object species is thepredominant species present (i.e., on a molar basis, more abundant thanany other individual macromolecular species in the composition), and asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50% (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition means that about 80% to 90% or more of the macromolecularspecies present in the composition is the purified species of interest.The object species is purified to essential homogeneity (contaminantspecies cannot be detected in the composition by conventional detectionmethods) if the composition consists essentially of a singlemacromolecular species. Solvent species, small molecules (<500 Daltons),stabilizers (e.g., BSA), and elemental ion species are not consideredmacromolecular species for purposes of this definition.

“Naturally-occurring” as applied to an object refers to the fact thatthe object can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory isnaturally-occurring.

A “host” refers to any animal including human or non-human animals, suchas rodents (e.g., mice or rats), primates, sheep, pigs, guinea pigs,etc.

“Treatment” refers to prophylactic treatment or therapeutic treatment.

A “prophylactic” treatment is a treatment administered to a host whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology.

A “therapeutic” treatment is a treatment administered to a host whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

I. Chimeric Proteins Comprising a Non-Toxic Pseudomonas Exotoxin aSequence and a Type IV Pilin Loop Sequence

In one aspect, the invention provides a chimeric protein comprising: anon-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loopsequence, the Type IV pilin loop sequence being located within thenon-toxic Pseudomonas exotoxin A sequence, wherein the chimeric proteinis capable of reducing the adhesion or adherence of a microorganismexpressing the Type IV pilin loop sequence to epithelial cells, andfurther wherein the chimeric protein, when introduced into a host, iscapable of generating polyclonal antisera that reduce adherence of themicroorganism expressing the Type IV pilin loop sequence to theepithelial cells. In some embodiments, the chimeric proteins of theinvention, when introduced into a host, are also capable of generatingpolyclonal antisera that neutralize cytotoxicity of Pseudomonas exotoxinA. In another aspect, the invention provides a chimeric proteincomprising: (a) a non-toxic Pseudomonas exotoxin A sequence comprisingdomain Ia, domain II, and domain III; and (b) a Type IV pilin loopsequence, wherein the Type IV pilin loop sequence is located betweendomain II and domain III of the non-toxic Pseudomonas exotoxin Asequence. In some embodiments, the chimeric protein comprises anon-toxic Pseudomonas exotoxin A sequence including domains Ia, II, andIII in the native organization structure, except that a Type IV pilinloop sequence, partially or completely, replaces domain Ib and islocated between domain II and domain III. Alternatively or additionally,in some embodiments, the chimeric protein comprises a Type IV pilin loopsequence in domain II, replacing amino acids 265 to 287. The nature ofnon-toxic Pseudomonas exotoxin A sequences, various domains of non-toxicPseudomonas exotoxin A sequences, Type IV pilin loop sequences, andtheir physical relationship within chimeric proteins of the inventionare described in detail below.

A. Non-toxic Pseudomonas Exotoxin A Sequences

As described in the Definition section above, Pseudomonas exotoxin A orPE is secreted by Pseudomonas aeruginosa and comprises three prominentdomains (Ia, II, and III) and one small subdomain (Ib) connectingdomains II and III. In nature, domain Ia of PE, spanning amino acids1-252, mediates cell binding. Domain II, spanning amino acids 253-364,mediates translocation of the protein to the cytosol. Domain Ib,spanning amino acids 365-399, has no known function. Domain III,spanning amino acids 400-613, is responsible for cytotoxicity andincludes an endoplasmic reticulum retention sequence. It also containssequences that mediates ADP ribosylation of elongation of factor 2(“EF2”), which inactivates protein synthesis and thus rendering PE to betoxic to cells. Thus, domain Ia or its variant that mediates cellbinding is referred to as “a cell recognition domain.” Domain II or itsvariant that mediates translocation of the proteins to the cytosol isreferred to as “a translocation domain.” Domain III or its variant thatfunctions in translocating the protein from the endosome to theendoplasmic reticulum is referred to as “an endoplasmic reticulumretention domain.”

A non-toxic Pseudomonas exotoxin A sequence refers to any Pseudomonasexotoxin A sequence that lacks ADP ribosylation activity. Generally, anon-toxic Pseudomonas exotoxin A sequence has one or more domains orportions of domains with certain biological activities. For example, anon-toxic Pseudomonas exotoxin A sequence may comprise a translocationdomain (e.g., domain II of Pseudomonas exotoxin A) and an endoplasmicreticulum domain (e.g., detoxified domain III of Pseudomonas exotoxin Awithout ADP ribosylation activity). In another example, a non-toxicPseudomonas exotoxin A sequence may be constructed by eliminating aminoacids 1-252 yielding a construct referred to as “PE40”. In anotherexample, a non-toxic Pseudomonas exotoxin A sequence may be constructedby eliminating amino acids 1-279 yielding a construct referred to as“PE37.” (See Pastan et al., U.S. Pat. No. 5,602,095.).

Optionally, a cell recognition domain of Pseudomonas exotoxin A (e.g.,domain I) or other cell recognition domains unrelated to Pseudomonasexotoxin A can be included in the present chimeric proteins. A cellrecognition domain can be linked, directly or indirectly, to the rest ofthe chimeric protein. For example, one can ligate sequences encoding acell recognition domain to the 5′ end of non-toxic versions of PE40 orPE37 constructs, which further comprise a Type IV pilin loop sequence.

1. Translocation Domain

The chimeric proteins of the invention comprise a non-toxic Pseudomonasexotoxin A sequence comprising a “PE translocation domain.” The PEtranslocation domain comprises an amino acid sequence sufficient toeffect translocation of chimeric proteins that have been endocytosed bythe cell into the cytosol. The amino acid sequence is identical to, orsubstantially identical to, a sequence selected from domain II of PE.

The amino acid sequence sufficient to effect translocation can bederived from the translocation domain of native PE. This domain spansamino acids 253-364. The translocation domain can include the entiresequence of domain II. However, the entire sequence is not necessary fortranslocation. For example, the amino acid sequence can minimallycontain, e.g., amino acids 280-344 of domain II of PE. Sequences outsidethis region, i.e., amino acids 253-279 and/or 345-364, can be eliminatedfrom the domain. This domain can also be engineered with substitutionsso long as translocation activity is retained.

The translocation domain functions as follows. After binding to areceptor on the cell surface, the chimeric proteins enter the cell byendocytosis through clathrin-coated pits. Residues 265 and 287 arecysteines that form a disulfide loop. Once internalized into endosomeshaving an acidic environment, the peptide is cleaved by the proteasefurin between Arg279 and Gly280. Then, the disulfide bond is reduced. Amutation at Arg279 inhibits proteolytic cleavage and subsequenttranslocation to the cytosol. Ogata et al., J. Biol. Chem. 265:20678-85(1990). However, a fragment of PE containing the sequence downstream ofArg279 (called “PE37”) retains substantial ability to translocate to thecytosol. Siegall et al., J. Biol. Chem. 264:14256-61 (1989). Sequencesin domain II beyond amino acid 345 also can be deleted withoutinhibiting translocation. Furthermore, amino acids at positions 339 and343 appear to be necessary for translocation. Siegall et al.,Biochemistry 30:7154-59 (1991).

Methods for determining the functionality of a translocation domain aredescribed below in the section on testing.

2. ER Retention Domain

The chimeric protein of the invention can also comprise an amino acidsequence encoding an “endoplasmic reticulum retention domain” as part ofa non-toxic exotoxin A sequence. The endoplasmic reticulum (“ER”)retention domain functions in translocating the chimeric protein fromthe endosome to the endoplasmic reticulum, from where it is transportedto the cytosol. The ER retention domain is located at the position ofdomain III in PE. The ER retention domain comprises an amino acidsequence that has, at its carboxy terminus, an ER retention sequence.The ER retention sequence in native PE is REDLK (SEQ ID NO:21). Lysinecan be eliminated (i.e., REDL (SEQ ID NO:22)) without a decrease inactivity. REDLK (from SEQ ID NO:21) can be replaced with other ERretention sequences, such as KDEL (SEQ ID NO:23), or polymers of thesesequences. See Ogata et al., J. Biol. Chem. 265:20678-85 (1990); Pastanet al., U.S. Pat. No. 5,458,878; Pastan et al., Annu. Rev. Biochem.61:331-54 (1992).

Sequences up-stream of the ER retention sequence can be the native PEdomain III (preferably de-toxified), can be entirely eliminated, or canbe replaced by another amino acid sequence. If replaced by another aminoacid sequence, the sequence can, itself, be highly immunogenic or can beslightly immunogenic. Activity of this domain can be assessed by testingfor translocation of the protein into the target cell cytosol using theassays described below.

In native PE, the ER retention sequence is located at the carboxyterminus of domain III. Domain III has two functions in PE. It exhibitsADP-ribosylating activity and directs endocytosed toxin into theendoplasmic reticulum. Eliminating the ER retention sequence from thechimeric protein does not alter the activity of Pseudomonas exotoxin asa superantigen, but does inhibit its utility to elicit an MHC ClassI-dependent cell-mediated immune response.

The ribosylating activity of PE is located between about amino acids 400and 600 of PE. In methods of vaccinating a host using the chimericproteins of this invention, it is preferable that the protein benon-toxic. One method of doing so is by eliminating ADP ribosylationactivity. In this way, the chimeric protein can function as a vector forType IV pilin loop sequences to be processed by the cell and presentedon the cell surface with MHC Class I molecules, rather than as a toxin.ADP ribosylation activity can be eliminated by, for example, deletingamino acid E553 (“ΔE553”) of the native PE. See, e.g., Lukac et al.,Infect. and Immun. 56:3095-3098 (1988). In another example, substitutionof histidine residue of PE at 426 with a tyrosine residue alsoinactivates the ADP-ribosylation of PE (see Kessler & Galloway, supra).Other amino acids in domain III can be modified from the protein toeliminate ADP ribosylation activity. An ER retention sequence isgenerally included at the carboxy-terminus of the chimeric protein.

In one embodiment, the sequence of the ER retention domain issubstantially identical to the native amino acid sequences of the domainIII, or a fragment of it. In some embodiments, the ER retention domainis domain III of PE.

In another embodiment, a cell recognition domain is inserted into theamino acid sequence of the ER retention domain (e.g., into domain III).For example, the cell recognition domain can be inserted just up-streamof the ER retention sequence, so that the ER retention sequence isconnected directly or within ten amino acids of the carboxy terminus ofthe cell recognition domain.

B. Cell Recognition Domain

Optionally, the chimeric protein of the invention can comprise an aminoacid sequence encoding a “cell recognition domain.” The cell recognitiondomain functions as a ligand for a cell surface receptor. It mediatesbinding of the protein to a cell. It can be used to target the chimericprotein to a cell which will transport it to the cytosol for processing.A cell recognition domain may not be necessarily included in thechimeric protein, as a Type IV pilin loop sequence within the chimericprotein targets receptors on epithelial cells.

The cell recognition domain functions to attach the chimeric protein toa target cell, and it can be any suitable material, e.g., a polypeptideknown to a particular receptor in the target cell. For example, the cellrecognition domain generally has the size of known polypeptide ligands,e.g., between about 10 amino acids and about 1500 amino acids, or about100 amino acids and about 300 amino acids. Several methods are usefulfor identifying functional cell recognition domains for use in chimericproteins. One method involves detecting binding between a chimericprotein that comprises the cell recognition domain with the receptor orwith a cell bearing the receptor. Other methods involve detecting entryof the chimeric protein into the cytosol, indicating that the firststep, cell binding, was successful. These methods are described indetail below in the section on testing.

In one embodiment, the cell recognition domain is domain Ia of PE,thereby targeting the chimeric protein to the α2-MR domain. In otherembodiments domain Ia can be substituted with ligands that bind to cellsurface receptors or antibodies or antibody fragments directed to cellsurface receptors. For example, to target epithelial cells, a cellbinding domain can be a ligand for or antibodies against the EGFreceptor, transferrin receptors, interleukin-2 receptors, interleukin-6receptors, interleukin-8 receptors, or Fc receptors, or poly-IgGreceptors. To target liver cells, a cell binding domain can be, e.g., aligand for or antibodies against asialoglycoprotein receptors. To targetT cells, a cell binding domain can be, e.g., a ligand for or antibodiesagainst CD3, CD4, CD8, or chemokine receptors. To target activatedT-cells and B-cells, a cell binding domain can be, e.g., a ligand for orantibodies against CD25. To target dendritic cells, a cell bindingdomain can be, e.g., ligands for or antibodies against CD11B, CD11C,CD80, and CD86 MHC class I and II. To target macrophages, a cell bindingdomain can be, e.g., ligands for or antibodies against TNFalphareceptors, chemokine receptors, TOLL receptors, M-CSF receptors, GM-CSFreceptors, scavenger receptors, and Fc receptors. To target endothelialcells, a cell binding domain can be, e.g., a ligand for or antibodiesagainst VEGF receptors. Also, cytokine receptors which are found in manycell types can be targeted. Pastan et al. Ann. Rev. Biochem. 61:331-54(1992).

The cell recognition domain can be located at any suitable positionwithin the present chimeric proteins. For example, the cell recognitiondomain can be located in the N-terminus of the chimeric protein (e.g.,position equivalent to domain Ia of non-toxic PE). However, this domaincan be moved out of the normal organizational sequence of exotoxin A.More particularly, the cell recognition domain can be inserted upstreamof the ER retention domain. Alternatively the cell recognition domaincan be chemically coupled to the rest of the chimeric protein. Also, thechimeric protein can include a first cell recognition domain at thelocation of the Ia domain and a second cell recognition domain upstreamof the ER retention domain. Such constructs can bind to more than onecell type. See, e.g., Kreitman et al., Bioconjugate Chem. 3:63-68(1992). For example, TGFa has been inserted into domain III just beforeamino acid 604, i.e., about ten amino acids from the carboxy-terminus.This chimeric protein binds to cells bearing EGF receptor. Pastan etal., U.S. Pat. No. 5,602,095.

The cell recognition domain can be inserted or attached to the rest ofthe chimeric proteins using any suitable methods. For example, thedomain can be attached to the rest of the chimeric protein directly orindirectly using a linker. The linker can form covalent bonds orhigh-affinity non-covalent bonds. Suitable linkers are well known tothose of ordinary skill in the art. In another example, the cellrecognition domain is expressed as a single chimeric polypeptide from anucleic acid sequence encoding the single contiguous chimeric protein.

C. Type IV Pilin Loop Sequences

The chimeric protein also comprises a Type IV pilin loop sequence withina non-toxic Pseudomonas exotoxin A sequence. The Type IV pilin loopsequence is generally derived from a sequence that forms an intrachaindisulfide loop at the C-terminus of the pilin protein. The Type IV pilinloop sequence allows the chimeric protein to react with asialoGM1receptors on epithelial cells. This loop is dominated by main chainresidues. Therefore, pilins from several strains bind the same receptordespite sequence variation and the difference in length (e.g., forcertain Pseudomonas strains, 12 and 17 amino acid loops (or 14 to 19amino acids including flanking cysteine residues)). A Type IV loop pilinsequence comprises at least about 5 amino acid residues, typicallybetween about 10 to 100 amino acids, more typically about 12 to 70 aminoacids, even more typically about 12 to 20 amino acids. Embodiments ofthe invention can have one unit of the Type IV pilin loop sequence ormultiple repeating units (e.g., 2, 3, 4, etc.) of the same or differentType IV pilin loop sequences. In some embodiments, the chimeric proteinscomprise more than one Type IV pilin loop sequences at differentlocations.

A Type IV pilin loop sequence can be derived from any microorganism thatadhere to epithelial cells. For example, a Type IV pilin sequence can bederived from bacteria or yeast, such as Pseudomonas aeruginosa,Neisseria meningtidis, Neisseria gonorrhoeae, Vibro cholera, Pasteurellamultocidam or Candida. Examples of a Type IV pilin sequence are shown asSEQ ID NOS: 3 to 20.

Type IV pilin sequences from different Pseudomonas aeruginosa strainsvary in terms of their sequence as well as their length. SeveralPseudomonas aeruginosa strains have a short pilin loop consisting of 14amino acids (from cysteine 129 to cysteine 142) as shown in Table 1below. Other Pseudomonas aeruginosa strains have a long pilin loopconsisting of 19 amino acids (from cysteine 133 to 151) as shown inTable 2 below. TABLE 1 P. aeruginosa strains Type IV pilin loop sequence(with a short pilin (Cysteine 129 to Cysteine loop) 142) PAKCTSDQDEQFIPKGC (SEQ ID NO: 3) T2A CTSTQDEMFIPKGC (SEQ ID NO: 4) PAO,90063 CKSTQDPMFTPKGC (SEQ ID NO: 5) CD, PA103 CTSTQEEMFIPKGC (SEQ ID NO:6) K122-4 CTSNADNKYLPKTC (SEQ ID NO: 7) KB7, 82932, 82935 CATTVDAKFRPNGC(SEQ ID NO: 8) 1071 CESTQDPMFTPKGC (SEQ ID NO: 9)

TABLE 2 P. aeruginosa strains (with a long pilin Type IV pilin loopsequence loop) (Cysteine 133 to Cysteine 151) 577B CNITKTPTAWKPNYAPANC(SEQ ID NO: 10) 1244, 9D2, P1 CKITKTPTAWKPNYAPANC (SEQ ID NO: 11) SBI-NCGITGSPTNWKANYAPANC (SEQ ID NO: 12)

Type IV pilin loop sequences from microorganisms other than P.aeruginosa can also be included in the chimeric proteins of theinvention. Examples of Type IV pilin loop amino acid sequences fromother microorganisms are shown in Table 3 below. TABLE 3 Micro- organismType IV pilin loop sequence Neisseria CGLPVARDDTDSATDVKADTTDNINTKHLPSTCmeningtidis (SEQ ID NO: 13) (Z49820) NeisseriaCGQPVTRGAGNAGKADDVTKAGNDNEKINTKHLPSTC meningtidis (SEQ ID NO: 14)(Z69262) Neisseria CGQPVTRAKADADAAGKDTTNIDTKHLPSTC meningtidis (SEQ IDNO: 15) (Z69261) Neisseria CGQPVTRTGDNDDTVADAKDGKEIDTKHLPSTC gonorrhoeae(SEQ ID NO: 16) (pilE; X66144) NeisseriaCGQPVKRDAGAKTGADDVKADGNNGINTKHLPSTC gonorrhoeae (SEQ ID NO: 17) (pilE;AF043648) Vibrio CKTLVTSVGDMFPFINVKEGAFAAVADLGDFETSVADA choleraATGAGVIKSIAPGSANLNLTNITHVEKLC (U09807) (SEQ ID NO: 18) VibrioCKTLITSVGDMFPYIAIKAGGAVALADLGDFENSAAAAE choleraTGVGVIKSIAPASKNLDLTNITHVEKLC (X64098) (SEQ ID NO: 19) PasteurellaCNGGSEVFPAGFC multocida (SEQ ID NO: 20) (AF154834)

One of skill in the art will recognize that the above described Type IVpilin sequences are merely exemplary and that other Type IV pilinsequences can be readily inserted into the chimeric proteins of thepresent invention. For example, Type IV pilin loop sequences describedin, e.g., U.S. Pat. No. 5,612,036 (Hodges et al.) can also beincorporated into the chimeric proteins of the present invention.

The Type IV pilin loop sequence can be located at any suitable positionwithin the chimeric protein of the invention. In one embodiment, theType IV pilin sequence is inserted between the translocation domain(e.g., domain II of non-toxic exotoxin A) and the ER retention domain(e.g., domain III of non-toxic exotoxin A). In another embodiment, thechimeric protein has the basic organization structure of non-toxicPseudomonas exotoxin A including domain Ia, domain II, domain lb, anddomain III, except that domain lb is, partially or completely, replacedby the Type IV pilin loop sequence. In native Pseudomonas exotoxin A,domain lb spans amino acids 365 to 399. The native lb domain isstructurally characterized by a disulfide bond between two cysteines atpositions 372 and 379. Domain lb is not essential for cell binding,translocation, ER retention or ADP ribosylation activity. Therefore, itcan be partially or entirely replaced by a Type IV pilin loop sequence.For example, a Type IV pilin loop sequence can be inserted between thetwo cysteines at positions 372 and 379, replacing the 6 amino acidresidues between the two cysteines. In. another embodiment, the Type IVpilin loop sequence can be inserted into the lb domain without removingany of the lb domain sequences. In another embodiment, the Type IV pilinloop sequence can be positioned in another location which forms acysteine-cysteine disulfide bonded loop, such as amino acids 265-287 ofdomain II of non-toxic Pseudomonas exotoxin A. In some embodiments, morethan one Type IV pilin loop sequences can be inserted into differentlocations within the chimeric protein.

Depending on whether the site of insertion within a non-toxicPseudomonas exotoxin A sequence has cysteine residues, a Type IV pilinloop sequence with or without cysteine residues at the N- and C-terminican be used. For example, if the site of insert in the non-toxicPseudomonas exotoxin A sequence does not have cysteine residues, then aType IV pilin loop sequence with cysteine residues at its termini (e.g.,14 amino acids shown in SEQ ID NO:3) can be inserted. In anotherexample, if a Type IV pilin loop sequence is inserted in thecysteine-cysteine loop of the native lb domain, replacing the six aminoacids between the cysteine residues, then a Type IV pilin loop sequencecan be a sequence without terminal cysteines (e.g., 12 amino acidsbetween the two cysteines shown in SEQ ID NO:3). Therefore, acysteine-cysteine loop can be preferably formed within the chimericprotein of the invention. When the Type IV pilin loop sequence withinthe chimeric protein is presented as a cysteine-cysteine disulfidebonded loop, the Type IV pilin loop structure may stick out from therest of the chimeric protein, where it is available to interact with,e.g., asialoGM1 receptors or with immune system components.

II. Chimeric Polynucleotides and Expression of Polynucleotides

A. Polynucleotides Encoding the Chimeric Proteins

In another aspect, the invention provides polynucleotides encoding thechimeric proteins of the invention. Suitable amino acid sequences ofnon-toxic Pseudomonas exotoxin A sequences (e.g., comprising atranslocation domain and an ER retention domain), cell recognitiondomains, and Type IV pilin loop sequences and their physical locationswithin the present chimeric proteins are described in detail above. Anypolynucleotides that encode these amino acid sequences are within thescope of the present invention.

1. Identification of Non-Toxic Pseudomonas Exotoxin A SequencesPolynucleotides that encode non-toxic Pseudomonas exotoxin A amino acidsequences may be identified, prepared and manipulated using any of avariety of well established techniques. A nucleotide encoding nativePseudomonas exotoxin A is shown as SEQ ID NO:1. The practitioner can usethis sequence to prepare non-toxic Pseudomonas exotoxin A sequencesusing various cloning and in vitro amplification methodologies known inthe art. PCR methods are described in, for example, U.S. Pat. No.4,683,195; Mullis et al. Cold Spring Harbor Symp. Quant. Biol. 51:263(1987); and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989);Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual (1995). Theseprimers can be used, e.g., to amplify either the full length sequence,partial sequences or a probe of one to several hundred nucleotides,which is then used to screen cDNA or genomic libraries for relatednucleic acid sequence homologs. Polynucleotides can also be isolated byscreening genomic or cDNA libraries (e.g., Pseudomonas aeruginosa) withprobes selected from the sequences of the desired polynucleotide understringent hybridization conditions.

As an illustration, to clone a Pseudomonas exotoxin A sequencecomprising all of the domains (domain Ia, domain II, domain lb, anddomain III), the following primers can be used:Forward—GGCCCATATGCACCTGATACCCCAT (SEQ ID NO:24); andReverse—GAATTCAGTTACTTCAGGTCCTCG (SEQ ID NO:25). To clone a Pseudomonasexotoxin A sequence comprising domain II, domain Ib, and domain III, thefollowing primers can be used: Forward—GGCCCATATGGAGGGCGGCAGCCTGGCC (SEQID NO:26); and Reverse—GAATTCAGTTACTTCAGGTCCTCG (SEQ ID NO:27).

Other Pseudomonas exotoxin A constructs that can be used in theembodiments of the invention are also described in, e.g., U.S. Pat. No.5,602,095 (Pastan et al.). As described in the '095 patent, eliminatingnucleotides encoding amino acids 1-252 yields a construct referred to as“PE40.” Eliminating nucleotides encoding amino acids 1-279 yields aconstruct referred to as “PE37.” Non-toxic versions of these constructs(which lack domain Ia of native exotoxin A) are particularly useful forligating them to sequences encoding heterologous cell recognitiondomains to the 5′ end of these constructs. These constructs canoptionally encode an amino-terminal methionine.

In addition, Pseudomonas exotoxin A can be further modified usingsite-directed mutagenesis or other techniques known in the art, to alterthe molecule for a particular desired application. Means to alterPseudomonas exotoxin A in a manner that does not substantially affectthe functional advantages provided by the PE molecules described hereincan also be used and such resulting molecules are intended to be coveredherein.

Non-toxic Pseudomonas exotoxin A sequences can be generated from thesePseudomonas exotoxin A sequences by modifying portions of domain III sothat they lack ADP ribosylation activity. The ribosylating activity ofPE is located between about amino acids 400 and 600 of nativePseudomonas exotoxin A. For example, deleting amino acid E553 (“ΔE553”)from domain III detoxifies the molecule. This detoxified PE is referredto as “PE ΔE553.” Other amino acids within domain III can be modifiedby, e.g., deletion, substitution or addition of amino acid residues, toeliminate ADP ribosylation activity. For example, substitution ofhistidine residue of PE at 426 with a tyrosine residue also inactivatesthe ADP-ribosylation of PE (see Kessler & Galloway, supra).

In some embodiments, non-toxic Pseudomonas exotoxin A sequences can befurther modified to accommodate cloning sites for insertion of a Type IVpilin loop sequence. For example, a cloning site for the Type IV pilinsequence can be introduced between the nucleotides encoding thecysteines of domain lb of non-toxic Pseudomonas exotoxin A. For example,a nucleotide sequence encoding a portion of the lb domain between thecysteine-encoding residues can be removed and replaced with a nucleotidesequence encoding an amino acid. sequence and that includes a PstIcloning site. This example is described in detail in the Examplesection. Alternatively, a longer portion of domain lb or entire domainlb can be removed and replaced with an amino acid sequence and thatincludes cloning site(s).

The construct can also be engineered to encode a secretory sequence atthe amino terminus of the protein. Such constructs are useful forproducing the chimeric proteins in mammalian cells. In vitro, suchconstructs simplify isolation of the chimeric proteins. In vivo, theconstructs are useful as polynucleotide vaccines; cells that incorporatethe construct will express the protein and secrete it where it caninteract with the immune system.

2. Identification Type IV Pilin Loop Sequences

Polynucleotides that encode Type IV pilin loop amino acid sequences maybe identified, prepared and manipulated using any of a variety ofwell-established techniques. Type IV pilin nucleotide and amino acidsequences from various microorganisms are well-known in the art. See,e.g., NCBI Database Accession No. M14849 J02609 for Pseudomonas PAKstrain; NCBI Database Accession No. AAC60462 for Pseudomonas T2A strain;NCBI Database Accession No. M11323 for Pseudomonas PAO strain; NCBIDatabase Accession No. P17837 for Pseudomonas CD strain; NCBI DatabaseAccession No. B31105 for Pseudomonas P1 strain; NCBI Database AccessionNo. Q53391 for Pseudomonas KB7 strain; NCBI Database Accession No.AAC60461 for Pseudomonas 577B strain; NCBI Database Accession No. A33105for Pseudomonas K122-4 strain; NCBI Database Accession Nos. Z49820,Z69262, and Z69261 for N. meningtidis; NCBI Database Accession Nos.X66144 and AF043648 for N. gonorrhoeae; NCBI Database Accession Nos.U09807 and X64098 for V. cholera; NCBI Database Accession No. AF154834for Pasteurella multocida. The practitioners can clone and identifyother pilin nucleotides and amino acid sequences from othermicroorganisms using various cloning and in vitro amplificationmethodologies known in the art. For example, to clone other pilin loopPseudomonas strains from a library, primers for amplification from thehighly conserved 5′ end of the pilin gene and the 3′ end of theneighboring gene (Nicotinate-nucleotide pyrophosphorylase) in thePseudomonas genome can be used. Exemplary primers PCR (listed in the 5′to 3′ direction) for sequencing the pilin genes are as follows: pilATG(26 nc) GAGATATTCATGAAAGCTCAAAAAGG (SEQ ID NO:28); and nadB4 (20 nc)ATCTCCATCGGCACCCTGAC (SEQ ID NO:29); or nadB 1 (21 nc)TGGAAGTGGAAGTGGAGAACC (SEQ ID NO:30).

From these Type IV pilin polynucleotides, the portion that forms theC-terminal intrachain disulfide loop (i.e., Type IV pilin loop) can bereadily identified visually. Examples of Type IV pilin loop amino acidsare shown as SEQ ID NO:3 to 20 in Tables 1-3 above. Any degeneratenucleotides encoding these and other Type IV pilin loop amino acids canbe used to construct chimeric polynucleotides of the invention. In someembodiments, to facilitate insertion of Type IV pilin loop sequence intoa non-toxic Pseudomonas exotoxin A sequence, 5′ and/or 3′ ends of TypeIV pilin loop nucleotide sequence can be modified to incorporatecohesive ends for cloning sites (e.g., PstI).

As described above, typically, a Type IV pilin loop sequence is insertedinto domain lb, or can partially or fully replace domain lb of non-toxicPseudomonas exotoxin A. In some embodiments, a Type IV pilin loopsequence can be inserted into other suitable locations within anon-toxic Pseudomonas exotoxin A sequence. For example, a Type IV pilinloop sequence can be inserted in another location of non-toxicPseudomonas exotoxin A which forms a cysteine-cysteine disulfide bondedloop, such as amino acids 265-287 of domain II of non-toxic Pseudomonasexotoxin A. Other suitable locations for insertion can be readily testedusing functional tests described herein. In some embodiments, more thanone Type IV pilin loop sequences can be inserted into chimericpolynucleotides of the invention (e.g., a first pilin loop sequence indomain Ib and a second pilin loop sequence in domain II).

3. Identification of Cell Recognition Domain

Polynucleotides encoding various cell recognition domains are well-knownin the art. As described above, in one embodiment, the cell recognitiondomain is domain Ia of PE, thereby targeting the chimeric protein to theα2-MR domain. In this embodiment, the cell recognition domain can bereadily included in the chimeric polynucleotides using SEQ ID NO:1 asdescribed above. In other embodiments domain Ia can be substituted withligands that bind to cell surface receptors or antibodies or antibodyfragments directed to cell surface receptors. Suitable ligands andantibodies or antibody fragments are described above in section IBabove. Suitable locations for insertion of cell recognition domain intochimeric proteins and chimeric polynucleotides are also described abovein section IB.

The cell recognition domain can be inserted or attached to the rest ofthe chimeric proteins using any suitable methods. For example, thedomain can be attached to the rest of the chimeric protein directly orindirectly using a linker. The linker can form covalent bonds orhigh-affinity non-covalent bonds. Suitable linkers are well known tothose of ordinary skill in the art. In another example, the cellrecognition domain is expressed as a single chimeric polypeptide from anucleic acid sequence encoding the single contiguous chimeric protein.

B. Expression Cassettes and Vectors

Embodiments of the invention also provide expression cassettes andvectors for expressing the present chimeric proteins. Expressioncassettes are recombinant polynucleotide molecules comprising expressioncontrol sequences operatively linked to a polynucleotide encoding thechimeric protein. Expression vectors comprise these expression cassettesin addition to other sequences necessary for replication in cells.

Expression vectors can be adapted for function in prokaryotes oreukaryotes by inclusion of appropriate promoters, replication sequences,markers, etc. for transcription and translation of mRNA. Theconstruction of expression vectors and the expression of genes intransfected cells involves the use of molecular cloning techniques alsowell known in the art. Sambrook et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,(Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc.). Useful promoters for suchpurposes include a metallothionein promoter, a constitutive adenovirusmajor late promoter, a dexamethasone-inducible MMTV promoter, a SV40promoter, a MRP polIII promoter, a constitutive MPSV promoter, atetracycline-inducible CMV promoter (such as the human immediate-earlyCMV promoter), and a constitutive CMV promoter. A plasmid useful forgene therapy can comprise other functional elements, such as selectablemarkers, identification regions, and other genes.

Expression vectors useful in this invention depend on their intendeduse. Such expression vectors must contain expression and replicationsignals compatible with the host cell. Expression vectors useful forexpressing the chimeric proteins include viral vectors such asretroviruses, adenoviruses and adeno-associated viruses, plasmidvectors, cosmids, and the like. Viral and plasmid vectors are preferredfor transfecting mammalian cells. The expression vector pcDNA1(Invitrogen, San Diego, Calif.), in which the expression controlsequence comprises the CMV promoter, provides good rates of transfectionand expression. Adeno-associated viral vectors are useful in the genetherapy methods of this invention.

A variety of means are available for delivering polynucleotides to cellsincluding, for example, direct uptake of the molecule by a cell fromsolution, facilitated uptake through lipofection (e.g., liposomes orimmunoliposomes), particle-mediated transfection, and intracellularexpression from an expression cassette having an expression controlsequence operably linked to a nucleotide sequence that encodes theinhibitory polynucleotide. See also Inouye et al., U.S. Pat. No.5,272,065; Methods in Enzymology, vol. 185, Academic Press, Inc., SanDiego, Calif. (D. V. Goeddel, ed.) (1990) or M. Krieger, Gene Transferand Expression—A Laboratory Manual, Stockton Press, New York, N.Y.,(1990). Recombinant DNA expression plasmids can also be used to preparethe polynucleotides of the invention for delivery by means other than bygene therapy, although it may be more economical to make shortoligonucleotides by in vitro chemical synthesis.

The construct can also contain a tag to simplify isolation of theprotein. For example, a polyhistidine tag of, e.g., six histidineresidues, can be incorporated at the amino terminal end of the protein.The polyhistidine tag allows convenient isolation of the protein in asingle step by nickel-chelate chromatography.

C. Recombinant Cells

The invention also provides recombinant cells comprising an expressioncassette or vectors for expression of the nucleotide sequences encodinga chimeric protein of this invention. Host cells can be selected forhigh levels of expression in order to purify the protein. The cells canbe prokaryotic cells, such as E. coli, or eukaryotic cells. Usefuleukaryotic cells include yeast and mammalian cells. The cell can be,e.g., a recombinant cell in culture or a cell in vivo.

E. coli has been successfully used to produce the chimeric proteins ofthe present invention. The protein can fold and disulfide bonds can formin this cell.

D. Chimeric Protein Purification and Preparation

Once a recombinant chimeric protein is expressed, it can be identifiedby assays based on the physical or functional properties of the product,including radioactive labeling of the product followed by analysis bygel electrophoresis, radioimmunoassay, ELISA, bioassays, etc.

Once the encoded protein is identified, it may be isolated and purifiedby standard methods including chromatography (e.g., high performanceliquid chromatography, ion exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. See,generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y.(1982), Deutscher, Methods in Enzymology Vol. 182: Guide to ProteinPurification, Academic Press, Inc. N.Y. (1990). The actual conditionsused will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, etc., and will be apparent to thosehaving skill in the art.

After biological expression or purification, the chimeric proteins maypossess a conformation substantially different than the nativeconformations of the constituent proteins. In this case, it is helpfulto denature and reduce the chimeric protein and then to cause theprotein to re-fold into the preferred conformation. Methods of reducingand denaturing polypeptides and inducing re-folding are well known tothose of skill in the art (see Debinski et al., J. Biol. Chem.268:14065-14070 (1993); Kreitman & Pastan, Bioconjug. Chem. 4:581-585(1993); and Buchner et al., Anal. Biochem. 205:263-270 (1992)). Debinskiet al., for example, describe the denaturation and reduction ofinclusion body polypeptides in guanidine-DTE. The polypeptide is thenrefolded in a redox buffer containing oxidized glutathione andL-arginine.

E. Testing Functional Properties of the Chimeric Protein

The functional properties of the chimeric protein as a whole or eachcomponent thereof are using various routine assays. For example, thechimeric proteins are tested in terms of cell recognition, cytosolictranslocation, Type IV pilin adhesion, and immunogenicity. The entirechimeric protein can be tested, or the function of various domains canbe tested by substituting them for native domains of the wild-typeexotoxin A.

1. Receptor Binding/Cell Recognition

To determine whether the cell binding domain present in the chimericprotein functions properly, the ability of the chimeric protein to bindto the target receptor (either isolated or on the cell surface) istested using various methods known in the art.

In one method, binding of the chimeric protein to a target is performedby affinity chromatography. For example, the chimeric protein isattached to a matrix in an affinity column, and binding of the receptorto the matrix detected. Alternatively, the target receptor is attachedto a matrix in an affinity column, and binding of the chimeric proteinto the matrix is detected.

Binding of the chimeric protein to receptors on cells can be tested by,for example, labeling the chimeric protein and detecting its binding tocells by, e.g., fluorescent cell sorting, autoradiography, etc.

In some embodiments, toxic version of chimeric proteins (which has ADPribosylation activity) can be used to test whether the cell bindingdomain of the chimeric proteins binds to its target receptor. Forexample, the toxic version of chimeric proteins can be incubated witheither cells that express the target receptors or cells that do notexpress the target receptors, and cytotoxic effects of the toxic versionof chimeric proteins can be determined (e.g., by measuring inhibition of[³H]leucine incorporation).

If antibodies have been identified that bind to the ligand from whichthe cell recognition domain is derived, they are also useful to detectthe existence of the cell recognition domain in the chimeric protein byimmunoassay, or by competition assay for the cognate receptor.

In above testing methods, typically a specific or selective reaction ofthe chimeric protein to a target will be at least twice backgroundsignal or noise and more typically more than 10 to 100 times background.

These methods are described in detail in, e.g., Kreitman et al., Proc.Natl. Acad. Sci. U.S.A 87:8291-5 (1990); Siegall et al., Semin. CancerBiol. 1:345-50 (1990); Siegall et al., Cancer Res. 50:7786-8 (1990);FitzGerald et al., J. Cell Biol. 126(6):1533-41 (1995).

2. Translocation to the Cytosol

To determine whether the translocation domain and the ER retentiondomain of the chimeric protein properly functions, the ability of thechimeric protein to gain access to the cytosol is tested.

a) Presence in the Cytosol

In one method, access to the cytosol is determined by detecting thephysical presence of the chimeric protein in the cytosol. For example,the chimeric protein can be labeled and the chimeric protein exposed tothe cell. Then, the cytosolic fraction is isolated and the amount oflabel in the fraction determined. Detecting label in the fractionindicates that the chimera has gained access to the cytosol. This resultcan be compared with a control, e.g., background noise or signal. If thedetectable label in the cytosolic fraction is at least twice backgroundsignal or noise and more typically more than 10 to 100 times background,then, this result indicates that the chimeric protein has gained accessto the cytosol.

b) ADP Ribosylation Activity

In another method, the ability of the translocation domain and ERretention domain to effect translocation to the cytosol can be testedwith a construct containing a domain III having ADP ribosylationactivity. Briefly, cells are seeded in tissue culture plates and exposedto the toxic version of the chimeric protein containing the modifiedtranslocation domain or ER retention sequence. ADP ribosylation activityis determined as a function of inhibition of protein synthesis by, e.g.,monitoring the incorporation of ³H-leucine. This method is furtherdescribed in detail in FitzGerald et al., J. Bio. Chem. 273:9951-9958(1998). The incorporation of ³H-leucine in cells exposed the toxicversion of the chimeric protein can be compared to that of a non-toxiccounterpart or to background noise. If the incorporation of ³H-leucinein cells exposed the toxic version is reduced by at least twice, moretypically more than 10 to 100 times that of the non-toxic counterpart(or compared to background noise), then it can be said that the chimericprotein has properly gained entry to the cytosol.

3. Type IV Pilin Loop Adhesion

If the Type IV pilin sequence within the chimeric protein has astructure that is exposed to a solvent and has near-native conformation,the Type IV pilin loop sequence within the chimeric protein should bindto, e.g., asialoGM1 receptors or other receptors on epithelial cells andalso compete with microorganisms expressing the Type IV pilin loopsequence for binding to these receptors. Therefore, whether or not theType IV pilin loop sequence is properly functioning within the chimericprotein is tested by measuring its ability to adhere to epithelial cellsor its ability to block adherence of microorganisms expressing a Type IVpilin loop sequence (e.g., P. aeruginosa) to epithelial cells. Theseassays can be readily designed by one of skill in the art.

As an example, if a Type IV pilin loop sequence is derived from P.aeruginosa or Candida, an adhesion assay can be performed with asubstrate coated with asialoGM1. Various concentrations of the chimericprotein comprising Type IV pilin sequence can be assayed for reactivitywith immobilized asialoGM1. To determine specificity of this reactivitybetween the chimeric protein and asialoGM1, a competition assay can beperformed. For example, soluble asialoGM1 can be added to interfere thechimeric protein binding to immobilized asialoGM1. This method isdescribed in detail in the example section IIB3. This binding result canbe compared to a control (e.g., the same chimeric protein without thepilin loop insert or with a scrambled pilin loop sequence insert). Ifthe amount of binding of the chimeric protein to immobilized asialo GM1is at least twice, typically about 10 to 100 times greater than thecontrol, then it can be said that the pilin loop insert in the chimericprotein is functioning properly.

In another example, one can test the ability of the chimeric protein toblock binding of microorganisms expressing the Type IV pilin loopsequence to epithelial cells. The selection of epithelial cells dependson which microorganism Type IV pilin loop sequence within the chimericprotein is derived from. For instance, if the Type IV pilin loopsequence within the chimeric protein is derived from V. cholera, thenintestinal epithelial cells can be used binding assays. If the Type IVpilin loop sequence within the chimeric protein is derived from N.gonorrhoeae, then epithelial cells of genital urinary system can be usedfor binding assays. If the Type IV pilin loop sequence within thechimeric protein is derived from P. aeruginosa, then lung epithelialcells can be used for binding assays.

As an illustration, various Pseudomonas aeruginosa strains that expressType IV pilin can be added different to the human lung epithelial cellline, A549, which will result in the binding of Pseudomonas aeruginosato these cells. Then, the chimeric protein can be added. If the Type IVpilin sequence within the chimeric protein is present in near-nativeconformation, the chimeric protein would compete with Pseudomonasaeruginosa binding and would result in reduction of Pseudomonasaeruginosa adherence to the epithelial cells. This method is describedin detail in the example section III below. The result from thiscompetition assay can be compared to the result obtained with a control(e.g., the same chimeric protein except without the pilin loop insert orthe same chimeric protein with a scrambled pilin loop sequence insert).If the chimeric protein can reduce Pseudomonas binding at least twice ortypically about 10 to 100 times better than the control, then it can besaid that the pilin loop insert in the chimeric protein is functioningproperly.

4. Immunogenicity

To determine whether the chimeric protein retains its immunogenicityrespect to both parts of the chimeric protein (i.e., a Type IV pilinloop sequence and a non-toxic Pseudomonas exotoxin A sequence),properties of the antisera raised against the chimeric protein aretested.

a) Immunogenicity of Type IV Pilin Sequence

Immunogenicity of a Type IV pilin sequence within the chimeric proteinis tested by adhesion test using the antisera raised against thechimeric protein. An animal, such as a mouse or a rabbit, can beimmunized with a composition comprising the chimeric protein asdescribed below in Example section IVA. The post immunization antiserafrom the animal can be obtained and prepared to determine if theantisera can inhibit binding of microorganisms expressing the Type IVpilin sequence to the epithelial cells. For example, Pseudomonasaeruginosa can be added to the epithelial cells, and the amount ofPseudomonas binding to the epithelial cells is determined. Then, thepost immunization antisera can be added to the epithelial cells todetermine if antisera reduce binding of Pseudomonas aeruginosa to theepithelial cells. This assay is described in detail in Example sectionIVB. If the pilin loop sequence within the chimeric protein is presentin near native conformation, then it is expected that antisera raisedagainst the chimeric protein (at a suitable dilution, e.g., 1:10 or1:100) can reduce Pseudomonas binding by at least about 20%, typicallyat least about 30%, more typically at least about 50%.

b) Toxin Neutralizing Response

Immunogenicity of a non-toxic Pseudomonas exotoxin A sequence within thechimeric protein is tested by using antisera raised against the chimericprotein. Specifically, post immunization antisera is tested for itsability to neutralize cytotoxicity of Pseudomonas exotoxin A. Forexample, one can test the inhibition of protein synthesis of purifiedPseudomonas exotoxin A on eukaryotic cells in culture. When Pseudomonasexotoxin A is added to eukaryotic cells, it reduces or prevents proteinsynthesis in cells, causing cell cytotoxicity. To determine if antiseracan reduce or inactivate cell cytotoxicity of Pseudomonas exotoxin A,Pseudomonas exotoxin A can be incubated with antisera containingantibodies directed against the chimeric protein. This incubated mixtureis added to cells in culture. Then, the effect of antisera on theprotein synthesis in the cells can be measured (e.g., monitoring theincorporation of [³H] leucine). This assay is described in Examplesection IVC below and also in Ogata et al., J. Biol. Chem.265(33):20678-85 (1990). If the non-toxic exotoxin A sequence within thechimeric protein is present in near-native conformation, then it isexpected that antisera raised against the chimeric protein (at asuitable dilution, e.g., 1:10 or 1:100) can reduce cytotoxicity ofPseudomonas exotoxin A by at least about 30%, typically at least about50%, more typically at least about 70%, 80%, 90%, 95%, or 99% comparedto a control (e.g., addition of purified Pseudomonas exotoxin A withoutantisera).

III. Compositions Comprising Chimeric Proteins or Polynucleotides

The invention also provides formulations of one or more chimericpolypeptide or polynucleotide compositions disclosed herein inpharmaceutically-acceptable solutions for administration to a cell or ananimal, either alone or in combination with other components.

A. Compositions Comprising Chimeric Proteins

The chimeric protein of the invention can be administered directly to asubject as a pharmaceutical composition. Administration is by any of theroutes normally used for introducing a chimeric protein into ultimatecontact with the tissue to be treated, preferably the mucosal membraneand epithelial cells. The compositions comprising chimeric proteins areadministered in any suitable manner, preferably with pharmaceuticallyacceptable carriers. Suitable methods of administering such modulatorsare available and well known to those of skill in the art. Although morethan one route can be used to administer a particular composition, aparticular route can often provide a more immediate and more effectivereaction than another route.

Pharmaceutical compositions comprising the chimeric proteins of theinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliarieswhich facilitate processing of the polypeptides into preparations whichcan be used pharmaceutically. Proper formulation is dependent upon theroute of administration chosen.

Pharmaceutically acceptable carriers, diluents, or excipients aredetermined in part by the particular composition being administered, aswell as by the particular method used to administer the composition.Accordingly, there are a wide variety of suitable formulations ofpharmaceutical compositions of the present invention. For example,pharmaceutical compositions can be formulated for topicaladministration, systemic formulations, injections, transmucosaladministration, oral administration, inhalation/nasal administration,rectal or vaginal administrations. Suitable formulations for variousadministration methods are described in, e.g., Remington'sPharmaceutical Sciences, 17^(th) ed. 1985.

Briefly, for topical administration, the proteins may be formulated assolutions, gels, ointments, creams, suspensions, etc. Systemicformulations include those designed for administration by injection,e.g. subcutaneous, intravenous, intramuscular, intrathecal orintraperitoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration. For injection, theproteins may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. For oral administration, a composition canbe readily formulated by combining the chimeric proteins withpharmaceutically acceptable carriers to enable the chimeric proteins tobe formulated as tablets, pills, capsules, liquids, gels, syrups,slurries, suspensions and the like. For administration by inhalation,the chimeric proteins for use according to the present invention areconveniently delivered in the form of an aerosol spray from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Theproteins may also be formulated in rectal or vaginal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

Other suitable formulations and administration methods will be readilyapparent to one of skill in the art and can be applied to the presentinvention.

B. Compositions Comprising Chimeric Polynucleotides

The invention also provides compositions comprising the polynucleotidesencoding the chimeric proteins (sometimes referred to as “chimericnucleic acids” or “chimeric polynucleotides”). These nucleic acids canbe inserted into any of a number of well-known vectors for thetransfection of target cells or host tissues. For example, nucleic acidsare delivered as DNA plasmids, naked nucleic acid, and nucleic acidcomplexed with a delivery vehicle such as a liposome. Viral vectordelivery systems include DNA and RNA viruses, which have either episomalor integrated genomes after delivery to the cell. For a review of genetherapy procedures, see Anderson, Science 256:808-813 (1992); Nabel &Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166(1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460(1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne,Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer &Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada etal., in Current Topics in Microbiology and Immunology Doerfler and Böhm(eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,microinjection, biolistics, virosomes, liposomes, immunoliposomes,polycation or lipid:nucleic acid conjugates, naked DNA, artificialvirions, and agent-enhanced uptake of DNA. Lipofection is described in,e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat.No. 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, WO 91/17424, WO 91/16024.Delivery can be to cells (ex vivo administration) or target tissues (invivo administration).

C. Vaccines

In some preferred embodiments of the present invention, vaccines areprovided. The vaccines will generally comprise one or morepharmaceutical compositions, such as those discussed above, incombination with an immunostimulant. An immunostimulant may be anysubstance that enhances or potentiates an immune response (antibodyand/or cell-mediated) to an exogenous antigen. Examples ofimmunostimulants include adjuvants, biodegradable microspheres (e.g.,polylactic galactide) and liposomes (into which the compound isincorporated; see, e.g., Fullerton, U.S. Pat. No. 4,235,877). Vaccinepreparation is generally described in, for example, Powell & Newman,eds., Vaccine Design (the subunit and adjuvant approach) (1995).Pharmaceutical compositions and vaccines within the scope of the presentinvention may also contain other compounds, which may be biologicallyactive or inactive.

Any of a variety of immunostimulants may be employed in the vaccines ofthis invention. For example, an adjuvant may be included. Most adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A. Suitable adjuvants arecommercially available as, for example, Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.); AS-2 and derivatives thereof(SmithKline Beecham, Philadelphia, Pa.); CWS, TDM, Leif, aluminum saltssuch as aluminum hydroxide gel (alum) or aluminum phosphate; salts ofcalcium, iron or zinc; an insoluble suspension of acylated tyrosine;acylated sugars; cationically or anionically derivatizedpolysaccharides; polyphosphazenes; biodegradable microspheres;monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF orinterleukin-2, -7, or -12, may also be used as adjuvants.

Any suitable carrier known in the art can be employed in the vaccines ofthe invention, and the type of carrier will vary depending on the modeof administration. The vaccines can be formulated for any appropriatemanner of administration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. These formulations and administrationmethods are described above, and will not be repeated in this section.

Pharmaceutical compositions and vaccines of the present invention may bepresented in unit-dose or multi-dose containers, such as sealed vials.Such containers are preferably hermetically sealed to preserve sterilityof the formulation until use. In general, formulations can be stored assuspensions, solutions or emulsions in oily or aqueous vehicles.Alternatively, a pharmaceutical composition or vaccine may be stored ina freeze-dried condition requiring only the addition of a sterile liquidcarrier immediately prior to use.

D. Effective Dose

Determination of an effective amount of the chimeric protein forinducing an immune response in a subject is well within the capabilitiesof those skilled in the art, especially in light of the detaileddisclosure provided herein.

An effective dose can be estimated initially from in vitro assays. Forexample, a dose can be formulated in animal models to achieve aninduction of an immune response using techniques that are well known inthe art. One having ordinary skill in the art could readily optimizeadministration to humans based on animal data. Dosage amount andinterval may be adjusted individually. For example, when used as avaccine, the polypeptides and/or polynucleotides of the invention may beadministered in about 1 to 3 doses for a 1-36 week period. Preferably, 3doses are administered, at intervals of about 3-4 months, and boostervaccinations may be given periodically thereafter. Alternate protocolsmay be appropriate for individual patients. A suitable dose is an amountof polypeptide or DNA that, when administered as described above, iscapable of raising an immune response in an immunized patient sufficientto protect the patient from infections by microorganisms expressing TypeIV pilin sequence for at least 1-2 years. In general, the amount ofpolypeptide or nucleic acid present in a dose (or produced in situ bythe DNA in a dose) ranges from about 1 pg to about 5 mg per kg host,typically from about 10 pg to about 1 mg, and preferably from about 100pg to about 1 μg. Suitable dose range will vary with the size of thepatient, but will typically range from about 0.1 mL to about 5 mL.

IV. Methods of Eliciting an Immune Response

The chimeric proteins of the invention are useful in eliciting an immuneresponse in a host. Eliciting a humoral immune response is useful in theproduction of antibodies that specifically recognize the Type IV pilinloop sequence or the non-toxic exotoxin A sequence and in immunizationagainst microorganisms that bear the Type IV pilin sequence.

A. Prophylactic and Therapeutic Treatments

The chimeric proteins can include the Type IV pilin loop sequences fromvarious pathogenic microorganisms, including Pseudomonas aeruginosa,Neisseria meningitides, Neisseria gonorrhoeae, Vibro cholera, etc.Accordingly, this invention provides prophylactic and therapeutictreatments for diseases involving the pathological activity of pathogensbearing the Type IV pilin loop sequences. The methods involve immunizinga subject with non-toxic Pseudomonas exotoxin A based chimeric proteinsbearing the Type IV pilin sequence. The resulting immune responses mountan attack against the pathogens, themselves. For example, if thepathology results from bacterial or yeast infection, the immune systemmounts a response against the pathogens.

B. Humoral Immune Response

The chimeric proteins are useful in eliciting the production ofantibodies against the Type IV loop pilin sequence and the non-toxicPseudomonas exotoxin A sequence by a subject. The chimeric proteins areattractive immunogens for making antibodies against the Type IV pilinloop sequences that naturally occur within a cysteine-cysteine loop:Because they contain the Type IV pilin loop sequences within acysteine-cysteine loop, they present the Type IV pilin loop sequence tothe immune system in near-native conformation. The resulting antibodiesgenerally recognize the native antigen better than those raised againstlinearized versions of the Type IV pilin sequence.

Methods for producing polyclonal antibodies are known to those of skillin the art. In brief, an immunogen, preferably a purified polypeptide, apolypeptide coupled to an appropriate carrier (e.g., GST, keyhole limpethemanocyanin, etc.), or a polypeptide incorporated into an immunizationvector, such as a recombinant vaccinia virus (see, U.S. Pat. No.4,722,848) is mixed with an adjuvant. Animals are immunized with themixture. An animal's immune response to the immunogenic preparation ismonitored by taking test bleeds and determining the titer of reactivityto the polypeptide of interest. When appropriately high titers ofantibody to the immunogen are obtained, blood is collected from theanimal and antisera are prepared. Further fractionation of the antiserato enrich for antibodies reactive to the polypeptide is performed wheredesired. See, e.g., Coligan, Current Protocols in ImmunologyWiley/Greene, NY (1991); and Harlow and Lane, Antibodies: A LaboratoryManual Cold Spring Harbor Press, NY (1989).

In various embodiments, the antibodies ultimately produced can bemonoclonal antibodies, humanized antibodies, chimeric antibodies orantibody fragments.

Monoclonal antibodies are prepared from cells secreting the desiredantibody. These antibodies are screened for binding to polypeptidescomprising the epitope, or screened for agonistic or antagonisticactivity, e.g., activity mediated through the agent comprising thenon-native epitope. In some instances, it is desirable to preparemonoclonal antibodies from various mammalian hosts, such as mice,rodents, primates, humans, etc. Description of techniques for preparingsuch monoclonal antibodies are found in, e.g., Stites et al. (eds.)Basic and Clinical Immunology (4th ed.) Lange Medical Publications, LosAltos, Calif., and references cited therein; Harlow and Lane, Supra;Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.)Academic Press, New York, N.Y.; and Kohler and Milstein (1975) Nature256: 495-497.

In another embodiment, the antibodies are humanized immunoglobulins.Humanized antibodies are made by linking the CDR regions of non-humanantibodies to human constant regions by recombinant DNA techniques. SeeQueen et al., U.S. Pat. No. 5,585,089.

In another embodiment of the invention, fragments of antibodies againstthe Type IV pilin loop sequence are provided. Typically, these fragmentsexhibit specific binding to the Type IV pilin loop sequence similar tothat of a complete immunoglobulin. Antibody fragments include separateheavy chains, light chains, Fab, Fab′F(ab′)₂ and Fv. Fragments areproduced by recombinant DNA techniques, or by enzymatic or chemicalseparation of intact immunoglobulins.

Other suitable techniques involve selection of libraries of recombinantantibodies in phage or similar vectors. See, Huse et al., Science 246:1275-1281 (1989); and Ward et al., Nature 341: 544-546 (1989).

An approach for isolating DNA sequences which encode a human monoclonalantibody or a binding fragment thereof is by screening a DNA libraryfrom human B cells according to the general protocol outlined by Huse etal., Science 246:1275-1281 (1989) and then cloning and amplifying thesequences which encode the antibody (or binding fragment) of the desiredspecificity. The protocol described by Huse is rendered more efficientin combination with phage display technology. See, e.g., Dower et al.,WO 91/17271 and McCafferty et al., WO 92/01047. Phage display technologycan also be used to mutagenize CDR regions of antibodies previouslyshown to have affinity for the polypeptides of this invention or theirligands. Antibodies having improved binding affinity are selected.

The antibodies of this invention are useful for affinity chromatographyin isolating agents bearing the Type IV pilin sequence. Columns areprepared, e.g., with the antibodies linked to a solid support, e.g.,particles, such as agarose, Sephadex, or the like, where a cell lysateis passed through the column, washed, and treated with increasingconcentrations of a mild denaturant, whereby purified agents arereleased.

As described in the Example section, sera from immunized rabbits had tworeactivities: one that blocks adhesion and one that neutralizes exotoxinA. Therefore, by introducing the chimeric protein as a composition(e.g., a vaccine) into a subject, antibodies that prevent colonizationof microorganisms bearing Type IV pilin sequences (e.g., Pseudomonasaeruginosa) can be provided in the subject. In particular forPseudomonas aeruginosa, should small numbers of these bacteria overcomethis defense, the normal destructive power of the exotoxin A will bealso neutralized by the antisera.

C. IgA-mediated Secretory Immune Response

Mucosal membranes are primary entryways for many infectious pathogens,including those bearing the Type IV pilin sequences. Mucosal membranesinclude, e.g., the mouth, nose, throat, lung, vagina, rectum and colon.As a defense against entry, the body secretes secretory IgA on thesurfaces of mucosal epithelial membranes against pathogens. Furthermore,antigens presented at one mucosal surface can trigger responses at othermucosal surfaces due to trafficking of antibody-secreting cells betweenthese mucosae. The structure of secretory IgA has been suggested to becrucial for its sustained residence and effective function at theluminal surface of a mucosa. As used herein, “secretory IgA” or “sIgA”refers to a polymeric molecule comprising two IgA immunoglobulins joinedby a J chain and further bound to a secretory component. While mucosaladministration of antigens can generate an IgG response, parenteraladministration of immunogens rarely produce strong sIgA responses.

Pseudomonas exotoxin binds to receptors on mucosal membranes. Therefore,the chimeric proteins comprising non-toxic exotoxin A sequences are anattractive vector for bringing the type IV pilin loop sequence to amucosal surface. There, the chimeric proteins elicit an IgA-mediatedimmune response against the chimeric proteins. Accordingly, thisinvention provides the non-toxic Pseudomonas exotoxin A-based chimericproteins comprising a Type IV pilin loop sequence from a pathogen thatgains entry through mucosal membranes. The cell recognition domain canbe targeted to any mucosal surface receptor. These chimeric proteins areuseful for eliciting an IgA-mediated secretory immune response againstimmunogens that gain entry to the body through mucosal surfaces. Thechimeric proteins used for this purpose should have ligands that bind toreceptors on mucosal membranes as their cell recognition domains. Forexample, epidermal growth factor binds to the epidermal growth factorreceptor on mucosal surfaces.

The chimeric proteins can be applied to the mucosal surface by any ofthe typical means, including pharmaceutical compositions in the form ofliquids or solids, e.g., sprays, ointments, suppositories or erodiblepolymers impregnated with the immunogen. Administration can involveapplying the immunogen to a plurality of different mucosal surfaces in aseries of immunizations, e.g., as booster immunizations. A boosterinoculation can also be administered parenterally, e.g., subcutaneously.The chimeric protein can be administered in doses of about 1 μg to 1000μg, e.g., about 10 μg to 100 μg.

The IgA response is strongest on mucosal surfaces exposed to theimmunogen. Therefore, in one embodiment, the immunogen is applied to amucosal surface that is likely to be a site of exposure to theparticular pathogen. Accordingly, depending on the site of exposure tothe particular pathogen, the chimeric proteins can be administered tothe lung, nasal mucosa, vaginal, anal or oral mucosal surfaces, or theycan be given as an oral medication. For example, for cystic fibrosispatients, the chimeric proteins can be administered to the lung.

Mucosal administration of the chimeric protein of this invention resultin strong memory responses, both for IgA and IgG. Therefore, invaccination with them, it is useful to provide booster doses eithermucosally or parenterally. The memory response can be elicited byadministering a booster dose more than a year after the initial dose.For example, a booster dose can be administered about 12, about 16,about 20 or about 24 months after the initial dose.

The potential value of a Pseudomonas vaccine relates in part to itsability to protect individuals broadly from the strains that are presentin the environment. Based on the length of the pilin loop insert, thereare two groupings for Ps. aeruginosa: one group with a 12 amino acidsequence and one with a 17 amino acid insert. Both loops apparently bindasialo-GM1 and are thought to exhibit similar structures. Reflectingthis, we note that our vaccine protein, containing a 12 amino acid loopfrom the PAK strain, was able to generate antibodies that were reactivenot only for strains with the shorter loop but also for the SBI-Nstrain, which displayed the longer loop. Our studies have also providedadditional sequence data for pilin and pilin loop sequences. We reporthere two pilin loop sequences (those for Ps. aeruginosa strain 1071 andPs. aeruginosa strain SBI-N) that have not previously been entered indatabases (Tables 1 and 2).

Chronic pulmonary colonization by Ps. aeruginosa is associated with adecline in the clinical course of CF patients. Frequently, antibiotictherapy, even via pulmonary delivery, fails to eradicate Ps. aeruginosainfections in these patients (Steinkamp, G., B. et al. Pediatr Pulmonol6(2):91-8(1989)). Controlling Ps. aeruginosa infections, or better yet,preventing them, has thus become a critical unmet medical need in thecare of CF patients ((Bauernfeind, A. et al. Behring Inst Mitt(98):256-61 (1997)). To address this, a number of vaccine approacheshave been explored, many focused on outer membrane constituents(Matthews-Greer, J. M., et al.; J Infect Dis 155(6):1282-91 (1987);Owen, P. Biochem Soc Trans 20(1):1-6 (1992); Sawa, et al.; Nat Med5(4):392-8(1999), some on toxins (Chen, T. Y., et al. J Biomed Sci6(5):357-63 (1999); Denis-Mize, K. S., et al.; FEMS Immunol MedMicrobiol 27(2):147-54 (2000); Gilleland, H. E., et al.; J Med Microbiol38(2):79-86 (1993); Matsumoto, et al.; J Med Microbiol 47(4):303-8(1988)). and some on a combination approach (Cryz, S. J., et al.;Antibiot Chemother 39:249-55 (1987); Cryz, S. J., et al. Infect Immun52(1):161-5 (1986); Cryz, S. J., et al.; Infect Immun 55(7):1547-51(1987); and Cryz, S. J., et al. J Infect Dis 154(4):682-8 (1986)(Johansen, H. K., et al.; APMIS 102(7):545-53(1994).

The compositions of the present invention in some embodiments are usedto treat persons at risk of infection and particularly, Pseudomonasaeruginosa infection. These persons include, in particular, hospitalizedpatients having cystic fibrosis, burn wounds, organ transplants,compromised immune function, or intravenous-drug addition.

Previously, we compared the subcutaneous route with mucosal delivery oftoxin-V3 loop proteins (Mrsny, R. et al., Vaccine 17(11-12):1425-33(1999). Results of mucosal vaccination indicated that a robust anti-V3loop response could be achieved with high titer responses of both serumIgG and secretory IgA antibodies. Because the toxin-pilin chimericprotein is a candidate vaccine to prevent Pseudomonas colonization inCF, one embodiment provides a vaccine delivered to target mucosalantibody responses at airway epithelia.

EXAMPLES

I. Construction of Plasmids

Four plasmids, pPE64, pPE64Δ553, pPE64pil, pPE64Δ553pil, wereconstructed. Plasmid pPE64 encodes native the Pseudomonas exotoxin A,except the plasmid encoded a slightly smaller version of PE that lackedmuch of domain lb and has a novel PstI site in domain lb as described indetail below. Plasmid pPE64Δ553 encodes the a non-toxic version ofplasmid pPE64, whereby the plasmid pPE64 was modified by subcloning tointroduce the enzymatically inactive domain III of PE (i.e., Glu atamino acid position 553 is deleted). To generate a PE-based pilinchimeric protein, an oligonucleotide duplex that encoded amino acids129-142 from the PAK strain of pilin was synthesized. Then plasmidpPE64pil is constructed based on plasmid pPE64, wherein a pilin loopsequence from P. aeruginosa PAK strain was inserted into the PstI siteof plasmid pPE64. Plasmid pPE64Δ553pil is constructed based on plasmidpPE64Δ553, wherein a pilin loop sequence from P. aeruginosa PAK strainwas inserted into the PstI site of plasmid pPE64Δ553. All of thesevectors were constructed without a bacterial secretion sequence whichallowed recombinant proteins to be expressed as inclusion bodies.

Specifically, plasmids pPE64 and pPE64Δ553 are constructed as follows.Plasmid pMOA1A2VK352 (Ogata et al., J. Biol. Chem. 267, 25396-401(1992)), encoding PE, was digested with Sfi1 and ApaI (residues 1143 and1275, respectively) and then re-ligated with a duplex containing a novelPstI site. The coding strand of the duplex had the following sequence:5′-tggccctgac cctggccgcc gccgagagcg agcgcttcgt ccggcagggc accggcaacgacgaggccgg cgcggcaaac ctgcagggcc-3′. The resulting plasmid encoded aslightly smaller version of PE and lacked much of domain lb. The PstIsite was then used to introduce duplexes encoding pilin loop sequencesflanked by cysteine residues. To make non-toxic proteins, vectors weremodified by the subcloning in an enzymatically inactive domain III frompVC45ΔE553. An additional subcloning, from pJH4 (Hwang et. al., Cell48:129-136 (1987)), was needed to produce a vector that lacked a signalsequence. Construction of plasmids pPE64 and pPE64Δ553 are alsodescribed in FitzGerald et al., J. Biol. Chem. 273(16):9951-8 (1998).

Plasmids pPE64pil and pPE64Δ553pil with a pilin loop sequence insertwere constructed based on plasmids pPE64 and pPE64Δ553, respectively. A54 bp sense oligonucleotide with cohesive ends for PstI and encoding the12 amino acid pilin loop of the PAK strain, was annealed with a 54 bpantisense oligonucleotide in 10 mM Tris/HCl, 50 mM NaCl pH 7.4. Thesense and antisense oligonucleotides had the following sequences: Sense5′-TTGTACTAGTGATCAGGATGAACAGTTTATTCCGAAAGGTTGTTCACGTATGCA-3′; Antisense5′-TACGTGAACAACCTTTCGGAATAAACTGTTCATCCTGATCACTAGTACAATGCA-3′. Annealingwas accomplished by heating to 94° C. for 5 min followed by cooling to25° C. over a period of 40 min. Plasmids pPE64 and pPE64Δ5532), encodingenzymatically active and inactive PE respectively, were digested withPstI at residue 1470. (FitzGerald, D. J., et al., J Biol Chem273(16):9951-8 (1998). Ligation with the phosphorylated pilinoligoduplex destroyed the PstI site and introduced a unique SpeI site. AXhoI/SpeI double digest was used to check for the correct orientation ofthe insert. Ligation of the pilin oligoduplex into the PstI-cut vectorwas followed by several characterization steps to confirm the presenceof the pilin insert in the correct orientation. Final constructs wereverified by dideoxy double strand sequencing.

II. Expression and Characterization of Proteins

A. Expression and Purification

Using the T7 expression system described by Studier et al., (MethodsEnzymol. 185:60-89 (1990)), four PE-related proteins were expressed E.coli. These included PE64, PE64Δ553, PE64pil and PE64Δ553pil.

Chimeric proteins were expressed and isolated as inclusion bodies asdescribed in Buchner et al., Anal. Biochem. 205(2):263-70 (1992). Eachprotein was expressed separately and purified to near homogeneity.Briefly, strain BL21(λDE3) was transformed with plasmids harboring a T7promoter upstream of the initial ATG of the toxin-expressing vectors.Cultures were grown in Superbroth (KD Medical, Bethesda, Md.) withampicillin (50 ug/ml) and then induced for protein expression by theaddition of IPTG (1 mM). After two hours of further culture, bacterialcells were harvested by centrifugation. Following cell lysis, expressedproteins were recovered in inclusion bodies.

Proteins were solubilized with Guanidine HCl (6.0 M), 2 mM EDTA pH 8.0plus dithioerythreitol (65 mM). Solubilized proteins were then refoldedby dilution into a redox shuffling buffer (Buchner et al., Anal.Biochem. 205(2):263-70 (1992). Refolded proteins were dialyzed against a20 mM Tris, 100 mM urea pH 7.4, adsorbed on Q Sepharose (AmershamPharmacia Biotech), washed with 150 mM NaCl, 20 mM Tris, 1 mM EDTA pH6.5 and eluted with 280 mM NaCl, 20 mM Tris, 1 mM EDTA. Eluted proteinswere diluted 5-fold and then adsorbed onto a MonoQ column (HR 10/10,Amersham Pharmacia Biotech) and further purified by the application of alinear salt gradient (0-0.4 M NaCl in Tris EDTA, pH 7.4). PE proteinseluted between 0.2 and 0.25 M NaCl. Final purification was achievedusing a gel filtration column (Superdex 200, Amersham Pharmacia Biotech)in PBS, pH 7.4.

B. Characterization of Proteins

1. Western Blot Analysis

The PK99H mouse monoclonal antibody and purified pilin proteins wereobtained from Dr. Randall Irvin, University of Alberta, Canada.Antimouse IgG and antirabbit IgG antibodies were used to detect primaryantibodies in Western blots and ELISAs (available from Jackson ImmunoResearch Lab, West Grove, Pa.).

Proteins were initially analyzed by SDS-PAGE (FIG. 2 A). Substantiallypure proteins were obtained using the purification scheme outlinedabove. In Western blot analysis the PE64pil and PE64Δ553pil proteinsreacted with PK99H, a monoclonal antibody to the C-terminal loop ofpilin (FIG. 2 B). The same antibody also reacted with solublepreparations of the same proteins, indicating that the pilin insert wasexposed on the surface of the PE-pilin chimeric protein. PE proteinswithout inserts did not react with the PK99H antibody (FIG. 2 B).

2. Cytotoxicity Assay

To investigate the influence of the pilin insert on toxin structure andfunction, the two enzymatically active proteins, PE64 and PE64pil, werecompared in a cytotoxicity assay. Cytotoxicity assay methods describedin Ogata et al., J. Biol. Chem. 265(33):20678-85 (1990) was used.Concentrations of PE64 or PE64pil ranging from 0.002-20 ng/ml were addedto L929 cells for an overnight incubation. Cytotoxicity was thendetermined by measuring the inhibition of cellular protein synthesis(e.g., monitoring the incorporation of ³H-leucine). Data indicated thatPE64 and PE64pil exhibited similar toxicities with IC₅₀ values in therange of 0.1 ng/ml for both proteins (FIG. 3). This result suggestedthat the insert of 14 amino acids did not unduly perturb toxin functionand, by inference, toxin structure.

3. Reactivity with Immobilized Asialo-GM1

Previous results had indicated that synthetic peptides derived from theC-terminus of pilin could block the binding of pili to epithelial cells(Irvin et al., Infect. Immun. 57(12):3720-6 (1989); Yu, L. et al., MolMicrobiol 19(5):1107-16 (1996)). Blocking was attributed to peptidebinding to asialo-GM1 on the surface of epithelial cells. To test thefunctionality of the pilin insert in the PE64 proteins, variousconcentrations of PE64pil were assayed for reactivity with immobilizedasialo-GM1.

96-well plates were coated with asialo-GM1 or monosialo-GM1 (Sigma ChemCo, St Louis, Mo.) that had been solubilized in methanol. A 100 μlsolution of ganglioside (5 μg/ml) was added to each well and evaporatedat 4° C. until dry. Wells were washed 3 times with PBS and blocked withFish-gelatin-PBS (BioFX, Randallstown, USA) for 16 h at 4° C. Testproteins in blocking buffer were added at various concentrations. Afterincubation for 1 h at 22° C., the supernatant was removed and boundprotein was detected using heat-inactivated anti PE64Δ553pil serum(1:100) as the primary antibody. For competition studies, proteins at0.2 ug/ml were incubated with 2 ug/ml of asialo-GM1 or monosialo-GM1 for30 min at room temperature. Samples were then added to asialo-GM1 coatedplates as above.

Increasing concentrations of PE64pil from 0.1-2.0 ug/ml reactedspecifically with immobilized asialo-GM1 (FIG. 4A). PE64 was used as acontrol and exhibited only a low level of binding (FIG. 4A). Additionalstudies were carried out to confirm the ganglioside specificity of bothPE64pil and PE64Δ553pil. Soluble asialo-GM1 reduced the binding ofPE64pil and PE64Δ553pil to immobilized asialo-GM1 while the addition ofmonosialo-GM1 did not (FIGS. 4B and 4C). Neither ganglioside interferedwith the low level binding of PE64 and PE64Δ553 (FIGS. 4B and 4C). Takentogether, these results confirmed not only the presence of reactivepilin sequences but revealed a gain-of-function for the PE64pilproteins.

III. Adhesion Assays

A. Pseudomonas Strains

The following strains of Pseudomonas were used in adhesion and otherassays: PAK, PAO1, SBN-1, 1071, M2, 82932, 82935 and 90063. Pseudomonasstrains used for adherence studies were grown on LB agar and then in M9minimal medium (KD Medical, Bethesda, Md.) supplemented with 0.4%glucose at 30° C. without shaking. Cultures in late log phase wereroutinely used for adhesion assays.

B. Cell Cultures

A549 (ATCC, CCL-185), L929 (ATCC, CCL-1), WI 38, Vero and CHO cells weremaintained in DMEM/F12 or RMPI 1640 supplemented with 10% fetal bovineserum (FBS), 2.5 mM glutamine, standard Penicillin/Streptomycin (100U/100 ug/ml, GibcoBRL, Grand Island, USA) (further designed as completemedium) in 5% CO₂ at 37° C. Cells were fed every 2 to 3 days andpassaged every 5 to 7 days. For assays, cells were seeded into 24-wellor 96-well plates and grown to confluence.

C. Quantification of Bacterial Adherence

To quantify the association of Pseudomonas with A549 cells, we followedthe adhesion assay described by Chi et al., Infect. Immun. 59(3):822-8(1991). Briefly, A549 cells were grown in a 24 well plates (antibioticfree medium), to a density of approximately 2×10⁴ cells per well. Cellswere washed three times in HBSS without serum and were overlayed with0.5 ml of DMEM/F12 complete medium without FBS. A MOI of 20 was achievedby adding 10 μl of an appropriate bacterial dilution. Plates wereincubated for 1 or 2 h at 37° C., 5% CO₂.

To remove unbound bacteria, cells were gently washed three times withHBSS. Cells were then fixed for 1 h in 3.7% paraformaldehyde, 200 mMHEPES, pH 7.2. Cells were washed twice with saline and stained with 10%Giemsa for 10 min. Samples were washed three times with water andexamined under light microscopy at 400× magnification. Adherent bacteriawere quantified by counting the cell-associated bacteria of one hundredA549 cells.

D. Results

Pilin-mediated adhesion to epithelial cells allows P. aeruginosa toinitiate an infection. Agents that block adherence will therefore reducethe bacterial burden. The following three peptides were synthesized: along C-terminal peptide (peptide 1: acetyl-KCTSDQDEQFIPKGCSK-NH₂)corresponding to amino acids 128-142 of the PAK strain (this peptide wasoxidized to allow the formation of a disulfide bond), a core peptide(peptide 2: acetyl-DEQFIPK-NH₂) corresponding to amino acids 134-140 anda scrambled peptide (peptide 3: acetyl-QIDPEFK-NH₂) having the sameamino acid composition as the core but in a jumbled sequence. To enhancestability, the N-termini of these synthetic peptides were acetylatedwhile the C-termini were amidated. These peptides were customsynthesized by Sigma Genosys. The same peptides were also synthesizedwith a biotin label.

To test these peptides functionally, an adhesion assay was devisedwhereby washed bacteria of P. aeruginosa PAK strain were added to thehuman lung epithelial cell line, A549. Specifically, cultures ofconfluent A549 cells were incubated 60 min at 37° C. with 40 μM peptide1, 40 μM peptide 2, 40 μM peptide 3, 2 nmol/ml PAK-pilin protein, 2nmol/ml PE64, 2 nmol/ml PE64Δ553, 2 nmol/ml PE64pil, 2 nmol/mlPE64Δ553pil and 4 nmol/ml bovine albumin. Washed once with prewarmedDMEM and P. aeruginosa PAK strain was added at a MOI of approximately 50in DMEM, 2% FBS. Bacteria were centrifuged onto the cells (700 g, 5 min)and incubated 60 min, 37° C. 5% CO₂. Adherence was determined asdescribed above.

The results were as follows. Adherence to A549 cells was reduced byapproximately 50% in the presence of 40 μM of the long or the core pilinpeptide (see FIG. 5). The scrambled peptide did not interfere withadherence.

Because the PE-pilin proteins had exhibited binding activity toasialo-GM1, these were also tested. At approximately the same molarconcentration as the synthetic peptides, PE64pil and PE64Δ553pil alsoblocked bacterial adherence. Effects were due to the presence of theinsert, because toxin molecules without insert failed to compete foradherence.

IV. Immune Response to PE64Δ553pil

A. Production of Polyclonal Antibodies

To test the ability of the toxin-pilin protein to generate relevantantibody responses, four rabbits were injected with the PE64Δ553pilprotein. Two rabbits (numbered 87 and 88) received the protein plusadjuvant (complete Freunds for the first injection followed byincomplete Freunds for subsequent injections) and two (numbered 89 and90) received the protein alone. Two hundred micrograms of protein perinjection was given subcutaneously for a total of four cycles spacedapproximately 2 weeks apart. About 12 ml serum was isolated biweeklyfrom each rabbit. The sera were heat inactivated to 20 min, 56° C. anddilutions thereof were used for assays without further purification.Anti-pilin titers were determined using an ELISA assay wherebiotinylated pilin peptides were immobilized on strepavidin coatedplates. Over the period of immunization, anti-pilin titers increased inall four animals (FIG. 6). However, the speed and extent of the responsewere greater in the two rabbits that received antigen plus adjuvant. Toavoid complement-mediated bacterial killing, immune sera were heatinactivated. This treatment did not significantly alter antibody titersin the ELISA assay (data not shown).

B. Inhibition of Adhesion by Post Immunization Sera

To assess antibody mediated inhibition of adherence, anti-PE64Δ553pilrabbit sera were incubated at dilutions from 1:20 to 1:100 with 4×10⁵bacteria at 22° C. for 30 min. Bacteria were then centrifuged,resuspended in DMEM without supplements and added to confluentmonolayers of A549 cells at a MOI of 20 for 1-2 hrs. Adherence wasdetermined as described above. Immune sera taken after the fourthinjection were compared to prebleed samples taken from the same rabbits.

1. Inhibition of P. aeruginosa (AK Strain)

Sera taken 2 weeks after the last injection were assayed for blockingactivity in the bacterial adherence assay. Compared to prebleeds, immunesera at various dilutions blocked adherence of the PAK strain of Ps.aeruginosa (FIG. 7A). Reduction of adherence ranged from 60% at adilution of 1:100 to 90% at a dilution of 1:20. At a dilution of 1:20,blocking activity was comparable without regard to the presence ofadjuvant in the antigen preparation (FIG. 7B).

2. Inhibition of P. aeruginosa (Various Strains)

Inhibition of PAK strain adhesion confirmed that rabbits responded tothe specific pilin sequence that was administered in the vaccine.However, because the C-terminal loop of pilin exhibits considerablesequence variation, it was important to determine the reactivity of theimmune sera for other strains of Ps. aeruginosa. Strains PAO1, 1071,SBI-N, 82935, 82932, 90063 1244 and M2 were tested for adherence to A549cells under similar conditions as the PAK strain. The specific cellbinding of all strains were reduced in adhesion when heat inactivatedimmune rabbit sera were mixed with bacteria at a 1:20 dilution (FIG.7C). The reduction in adhesion among the different strains was more orless in the range of the PAK strain (about 90% reduction).

While it was unlikely that each of the above strains expressed the sameloop sequence as the PAK strain, it was of interest to analyzevariations at this portion of the pilin gene. Pilin sequences weredetermined by generating PCR clones of each strain's pilin gene andsequencing these. Primers for amplification were from the 5′ end of thepilin gene and the 3′ end of the neighboring gene (Nicotinate-nucleotidepyrophosphorylase) in the Pseudomonas genome (to be described in greaterdetail elsewhere). Results revealed the following: most strainsexhibited a 12 amino acid loop while one, SBI-N, had a 17 amino acidloop. Strains 82932 and 82935 had the same loop sequence as KB7(accession No, Q53391) and 90063 had a loop that matched PAO1 (accessionNo, A25023). Strains 1071 and SBI-N exhibited loops with novel sequences(See Tables 1 and 2). Strain M2, a mouse isolate, was not sequenced.

B. Toxin Neutralizing Response

The inhibition of protein synthesis of purified PE64 and PE64pil oneukaryotic cells in culture was determined as described in Ogata et al.,J. Biol. Chem. 265(33):20678-85 (1990). For inactivating cytotoxicactivity, the PE64pil proteins were incubated 30 min at 22° C. withrabbit sera, containing anti PE64Δ553pil antibodies, prior they wereadded to L929 or A549 cells in 24 well tissue culture dishes.

Rabbit antisera were evaluated for toxin neutralizing activity. All fourof the immunized rabbits at a 1:20 dilution of sera neutralized 1.0μg/ml of toxin completely (FIG. 8). From these results, it was concludedthat PE-pilin vaccine can generate antibodies of two reactivities: onethat blocks adhesion and one that neutralizes the exotoxin.

The present invention provides novel materials and methods for chimericproteins comprising a non-toxic Pseudomonas exotoxin A and a Type IVpilin loop sequence. While specific examples have been provided, theabove description is illustrative and not restrictive. Any one or moreof the features of the previously described embodiments can be combinedin any manner with one or more features of any other embodiments in thepresent invention. Furthermore, many variations of the invention willbecome apparent to those skilled in the art upon review of thespecification. The scope of the invention should, therefore, bedetermined not with reference to the above description, but insteadshould be determined with reference to the appended claims along withtheir full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, Applicants do not admit any particular reference is “priorart” to their invention.

1. A chimeric protein comprising: a non-toxic Pseudomonas exotoxin Asequence and a Type IV pilin loop sequence, the Type IV pilin loopsequence being located within the non-toxic Pseudomonas exotoxin Asequence, wherein the chimeric protein is capable of reducing adherenceof a microorganism expressing the Type IV pilin loop sequence toepithelial cells, and further wherein the chimeric protein, whenintroduced into a host, is capable of generating polyclonal antiserathat reduce adherence of the microorganism expressing the Type IV pilinloop sequence to the epithelial cells.
 2. The chimeric protein of claim1, wherein the chimeric protein, when introduced into the host, is alsocapable of generating polyclonal antisera that neutralize cytotoxicityof Pseudomonas exotoxin A.
 3. The chimeric protein of claim 1, whereinthe non-toxic Pseudomonas exotoxin A sequence comprises: (a) atranslocation domain sufficient to effect translocation of the chimericprotein to a cell cytosol; and (b) an endoplasmic reticulum retentiondomain that functions to translocate the chimeric protein from endosometo endoplasmic reticulum.
 4. The chimeric protein of claim 3, whereinthe chimeric protein further comprises a cell recognition domain thatfunctions as a ligand for a cell surface receptor and that mediatesbinding of the chimeric protein to a cell.
 5. The chimeric protein ofclaim 4, wherein the Type IV pilin loop sequence is located between thetranslocation domain and the endoplasmic reticulum retention domain. 6.The chimeric protein of claim 5, wherein the Type IV pilin loop sequencecomprises cysteine residues at both the N- and C-termini of the Type IVpilin loop sequence.
 7. The chimeric protein of claim 5, wherein theType IV pilin loop sequence is from bacteria or yeast.
 8. The chimericprotein of claim 7, wherein the Type IV pilin loop sequence is fromPseudomonas aeruginosa, Neisseria meningtidis, Neisseria gonorrhoeae,Vibro cholera, Pasteurella multocidam, or Candida.
 9. The chimericprotein of claim 8, wherein the Type IV pilin loop sequence is fromPseudomonas aeruginosa.
 10. The chimeric protein of claim 9, wherein theType IV pilin loop sequence is selected from the group consisting of SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, and SEQ ID NO:10.
 11. The chimeric protein of claim5, wherein the translocation domain comprises amino acids 280 to 364 ofdomain II of Pseudomonas exotoxin A.
 12. The chimeric protein of claim5, wherein the translocation domain is domain II of Pseudomonas exotoxinA.
 13. The chimeric protein of claim 5, wherein the endoplasmicreticulum retention domain is domain III of Pseudomonas exotoxin Aexcept that amino acid Glu at position of 553 is deleted.
 14. Thechimeric protein of claim 1, wherein the chimeric protein comprises morethan one Type IV pilin loop sequence.
 15. The chimeric protein of claim5, wherein the cell recognition domain is domain Ia of Pseudomonasexotoxin A.
 16. The chimeric protein of claim 5, wherein the cellrecognition domain binds to α2-macroglobulin receptor, epidermal growthfactor receptor, transferrin receptor, interleukin-2 receptor,interleukin-6 receptor, interleukin-8 receptor, Fc receptor, poly-IgGreceptor, asialoglycoprotein receptor, CD3, CD4, CD8, chemokinereceptor, CD25, CD11B, CD11C, CD80, CD86, TNFalpha receptor, TOLLreceptor, M-CSF receptor, GM-CSF receptor, scavenger receptor, VEGFreceptor, or cytokine receptor.
 17. A chimeric protein comprising: (a) anon-toxic Pseudomonas exotoxin A sequence comprising domain Ia, domainII, and domain III; and (b) a Type IV pilin loop sequence, wherein theType IV pilin loop sequence is located between domain II and domain IIIof the non-toxic Pseudomonas exotoxin A sequence.
 18. The chimericprotein of claim 17, wherein the non-toxic Pseudomonas exotoxin Asequence has the amino acid sequence of SEQ ID NO:2 with ΔE553.
 19. Thechimeric protein of claim 17, wherein the Type IV pilin loop sequence isfrom Pseudomonas aeruginosa, Neisseria meningtidis, Neisseriagonorrhoeae, Vibro cholera, Pasteurella multocidam, or Candida.
 20. Thechimeric protein of claim 17, wherein the Type IV pilin loop sequence isfrom Pseudomonas aeruginosa.
 21. A polynucleotide encoding a chimericprotein, the chimeric protein comprising: a non-toxic Pseudomonasexotoxin A sequence and a Type IV pilin loop sequence, the Type IV pilinloop sequence being located within the non-toxic Pseudomonas exotoxin Asequence, wherein the chimeric protein is capable of reducing adherenceof a microorganism expressing the Type IV pilin loop sequence toepithelial cells, and further wherein the chimeric protein, whenintroduced into a host, is capable of generating polyclonal antiserathat prevent adherence of the microorganism expressing the Type IV pilinloop sequence to the epithelial cells.
 22. The polynucleotide of claim21, wherein the chimeric protein, when introduced into the host, is alsocapable of generating polyclonal antisera that neutralize cytotoxicityof Pseudomonas exotoxin A.
 23. The polynucleotide of claim 21, whereinthe non-toxic Pseudomonas exotoxin A sequence comprises: (a) atranslocation domain sufficient to effect translocation of the chimericprotein to a cell cytosol; and (b) an endoplasmic reticulum retentiondomain that functions to translocate the chimeric protein from endosometo endoplasmic reticulum.
 24. The polynucleotide of claim 23, whereinthe chimeric protein further comprises a cell recognition domain thatfunctions as a ligand for a cell surface receptor and that mediatesbinding of the chimeric protein to a cell.
 25. The polynucleotide ofclaim 24, wherein the Type IV pilin loop sequence is located between thetranslocation domain and the endoplasmic reticulum retention domain. 26.The polynucleotide of claim 25, wherein the Type IV pilin loop sequencecomprises cysteine residues at both the N- and C-termini of the Type IVpilin loop sequence.
 27. The polynucleotide of claim 25, wherein theType IV pilin loop sequence is from bacteria or yeast.
 28. Thepolynucleotide of claim 27, wherein the Type IV pilin loop sequence isfrom Pseudomonas aeruginosa, Neisseria meningtidis, Neisseriagonorrhoeae, Vibro cholera, Pasteurella multocidam, or Candida.
 29. Thepolynucleotide of claim 28, wherein the Type IV pilin loop sequence isfrom Pseudomonas aeruginosa.
 30. The polynucleotide of claim 29, whereinthe Type IV pilin loop sequence is selected from the group consisting ofSEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, and SEQ ID NO:10.
 31. The polynucleotide of claim 25,wherein the translocation domain comprises amino acids 280 to 364 ofdomain II of Pseudomonas exotoxin A.
 32. The polynucleotide of claim 25,wherein the translocation domain is domain II of Pseudomonas exotoxin A.33. The polynucleotide of claim 25, wherein the endoplasmic reticulumretention domain is domain III of Pseudomonas exotoxin A except thatamino acid Glu at position of 553 is deleted.
 34. The polynucleotide ofclaim 25, wherein the cell recognition domain is domain Ia ofPseudomonas exotoxin A.
 35. The polynucleotide of claim 25, wherein thecell recognition domain binds to α2-macroglobulin receptor, epidermalgrowth factor receptor, transferring receptor, Fc receptor, poly-IgGreceptor, asialoglycoprotein receptor, CD3, CD4, CD8, chemokinereceptor, CD25, CD11B, CD11C, CD80, CD86, TNFalpha receptor, TOLLreceptor, M-CSF receptor, GM-CSF receptor, scavenger receptor, VEGFreceptor, or cytokine receptor.
 36. A polynucleotide encoding a chimericprotein, the chimeric protein comprising: (a) a non-toxic Pseudomonasexotoxin A sequence comprising domain Ia, domain II, and domain III; and(b) a Type IV pilin loop sequence, wherein the Type IV pilin loopsequence is located between domain II and domain III of the non-toxicPseudomonas exotoxin A sequence.
 37. The polynucleotide of claim 36,wherein the non-toxic Pseudomonas exotoxin A sequence has the amino acidsequence of SEQ ID NO:2 with ΔE553.
 38. The polynucleotide of claim 36,wherein the Type IV pilin loop sequence is from Pseudomonas aeruginosa,Neisseria meningtidis, Neisseria gonorrhoeae, Vibro cholera, Pasteurellamultocidam, or Candida.
 39. The polynucleotide of claim 36, wherein theType IV pilin loop sequence is from Pseudomonas aeruginosa.
 40. Anexpression cassette comprising the polynucleotide of claim
 21. 41. Acell comprising the expression cassette of claim
 40. 42. A compositioncomprising a chimeric protein, the chimeric protein comprising: anon-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loopsequence, the Type IV pilin loop sequence being located within thenon-toxic Pseudomonas exotoxin A sequence, wherein the chimeric proteinis capable of reducing adherence of a microorganism expressing the TypeIV pilin loop sequence to epithelial cells, and further wherein thechimeric protein, when introduced into a host, is capable of generatingpolyclonal antisera that prevent adherence of the microorganismexpressing the Type IV pilin loop sequence to the epithelial cells. 43.The composition of claim 42, wherein the chimeric protein, whenintroduced into the host, is also capable of generating polyclonalantisera that neutralize cytotoxicity of Pseudomonas exotoxin A.
 44. Thecomposition of claim 42, wherein the composition further comprises apharmacologically acceptable carrier.
 45. The composition of claim 42,wherein the composition is formulated as a nasal or oral spray.
 46. Thecomposition of claim 42, wherein the non-toxic Pseudomonas exotoxin Asequence comprises: (a) a translocation domain sufficient to effecttranslocation of the chimeric protein to a cell cytosol; and (b) anendoplasmic reticulum retention domain that functions to translocate thechimeric protein from endosome to endoplasmic reticulum.
 47. Thecomposition of claim 46, wherein the chimeric protein further comprisesa cell recognition domain that functions as a ligand for a cell surfacereceptor and that mediates binding of the chimeric protein to a cell.48. The composition of claim 47, wherein the Type IV pilin loop sequenceis from Pseudomonas aeruginosa.
 49. A method for eliciting an immuneresponse in a host, the method comprising the step of administering tothe host an immunologically effective amount of a composition comprisinga chimeric protein comprising: a non-toxic Pseudomonas exotoxin Asequence and a Type IV pilin loop sequence, the Type IV pilin loopsequence being located within the non-toxic Pseudomonas exotoxin Asequence, wherein the chimeric protein is capable of reducing adherenceof a microorganism expressing the Type IV pilin loop sequence toepithelial cells, and further wherein the chimeric protein, whenintroduced into the host, is capable of generating polyclonal antiserathat prevent adherence of the microorganism expressing the Type IV pilinloop sequence to the epithelial cells.
 50. The method of claim 49,wherein the chimeric protein, when introduced into the host, is capableof generating polyclonal antisera that neutralize cytotoxicity ofPseudomonas exotoxin A.
 51. The method of claim 49, wherein the host isa human.
 52. The method of claim 49, wherein the non-toxic Pseudomonasexotoxin A sequence comprises: (a) a translocation domain sufficient toeffect translocation of the chimeric protein to a cell cytosol; and (b)an endoplasmic reticulum retention domain that functions to translocatethe chimeric protein from endosome to endoplasmic reticulum.
 53. Themethod of claim 52, wherein the chimeric protein further comprises acell recognition domain that functions as a ligand for a cell surfacereceptor and that mediates binding of the chimeric protein to a cell.54. The method of claim 53, wherein the Type IV pilin loop sequence isfrom Pseudomonas aeruginosa.
 55. A method of eliciting an immuneresponse in a host, the method comprising the step of administering tothe host an immunologically effective amount of an expression cassettecomprising a polynucleotide encoding a chimeric protein comprising: anon-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loopsequence, the Type IV pilin loop sequence being located within thenon-toxic Pseudomonas exotoxin A, wherein the chimeric protein iscapable of reducing adherence of a microorganism expressing the Type IVpilin loop sequence to epithelial cells, and further wherein thechimeric protein, when introduced into the host, is capable ofgenerating polyclonal antisera that reduce adherence of themicroorganism expressing the Type IV pilin loop sequence to theepithelial cells.
 56. The method of claim 55, wherein the chimericprotein, when introduced into the host, is capable of generatingpolyclonal antisera that neutralize cytotoxicity of Pseudomonas exotoxinA.
 57. The method of claim 55, wherein the host is a human.
 58. Themethod of claim 55, wherein the non-toxic Pseudomonas exotoxin Asequence comprises: (a) a translocation domain sufficient to effecttranslocation of the chimeric protein to a cell cytosol; and (b) anendoplasmic reticulum retention domain that functions to translocate thechimeric protein from endosome to endoplasmic reticulum.
 59. The methodof claim 58, wherein the chimeric protein further comprises a cellrecognition domain that functions as a ligand for a cell surfacereceptor and that mediates binding of the chimeric protein to a cell.60. The method of claim 59, wherein the Type IV pilin loop sequence isfrom Pseudomonas aeruginosa.
 61. A method of generating antibodiesspecific for a Type IV pilin loop sequence, comprising introducing intoa host a composition comprising a chimeric protein comprising anon-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loopsequence, the Type IV pilin loop sequence being located within thenon-toxic Pseudomonas exotoxin A, wherein the chimeric protein iscapable of reducing adherence of a microorganism expressing the Type IVpilin loop sequence to epithelial cells, and further wherein thechimeric protein, when introduced into the host, is capable ofgenerating polyclonal antisera that reduce adherence of themicroorganism expressing the Type IV pilin loop sequence to epithelialcells.
 62. The method of claim 61, wherein the chimeric protein, whenintroduced into the host, is capable of generating polyclonal antiserathat neutralize cytotoxicity of Pseudomonas exotoxin A.
 63. The methodof claim 61, wherein the host is a human.
 64. The method of claim 61,wherein the non-toxic Pseudomonas exotoxin A sequence comprises: (a) atranslocation domain sufficient to effect translocation of the chimericprotein to a cell cytosol; and (b) an endoplasmic reticulum retentiondomain that functions to translocate the chimeric protein from endosometo endoplasmic reticulum.
 65. The method of claim 64, wherein thechimeric protein further comprises a cell recognition domain thatfunctions as a ligand for a cell surface receptor and that mediatesbinding of the chimeric protein to a cell.
 66. The method of claim 65,wherein the Type IV pilin loop sequence is from Pseudomonas aeruginosa.